Herbicide-tolerant plants through bypassing metabolic pathway

ABSTRACT

DNA sequence of a gene of hydroxy-phenyl pyruvate dioxygenase and production of plants containing a gene of hydroxy-phenyl pyruvate dioxygenase and which are resistant to herbicides. DNA sequence of a gene of hydroxy-phenyl pyruvate dioxygenase; isolation from a bacteria or a plant; utilization for obtaining plants tolerant to herbicides.

SUBMISSION ON COMPACT DISC

The contents of the following submission on compact discs areincorporated herein by reference in its entirety: two copies of theSequence Listing (COPY 1 and COPY 2) and a computer readable form copyof the Sequence Listing (CRF COPY), all on compact disc, eachcontaining: file name: 5500-118 Sequence Listing II, date recorded: Dec.5, 2006, size: 158 KB.

The present invention relates to a novel method for making plantstolerant to herbicides, in particular to HPPD-inhibiting herbicides, tothe nucleic acid sequences encoding enzymes which can be used in thismethod, to the expression cassettes containing them and to thetransgenic plants comprising at least one of these expression cassettes.

Hydroxyphenylpyruvate dioxygenases are enzymes which catalyze thereaction of conversion of para-hydroxyphenylpyruvate (HPP) tohomogentisate. This reaction takes place in the presence of iron (Fe²⁺)in the presence of oxygen (N. P. Crouch et al., Tetrahedron, 53, 20,6993-7010, 1997).

Certain molecules which inhibit this enzyme are, moreover, known, whichattach to the enzyme so as to inhibit the conversion of HPP tohomogentisate. Some of these molecules have found a use as herbicides,insofar as inhibition of the reaction in plants leads to a bleaching ofthe leaves of the treated plants and to the death of said plants (K. E.Pallett et al., 1997 Pestic. Sci. 50 83-84). Such herbicides having HPPDas the target, described in the state of the art, are in particularisoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482,EP 682,659, U.S. Pat. No. 5,424,276), in particular isoxaflutole, aherbicide selective for maize, diketonitriles (EP 496 630, EP 496 631),in particular 2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-CF₃phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-2,3-Cl₂phenyl))propane-1,3-dione,triketones (EP 625 505, EP 625 508, U.S. Pat. No. 5,506,195), inparticular sulcotrione or mesotrione, or else pyrazolinates.

Assays to confirm that HPPD is indeed the target for diketonitriles(DKNS) and to demonstrate that HPPD is, at least at certain doses, theonly target for diketonitriles, were carried out in the laboratory bygerminating Arabidopsis seeds on three types of medium under sterileconditions in vitro:

-   1 Murashige and Skoog medium (T. Murashige and F. Skoog, 1962. A    revised medium for a rapid growth and bioassays with tobacco tissue    culture. Physiol. Plant. 15, 473-479), control experiment for    germination-   2 MS medium plus DKN at a dose of 1 ppm-   3 MS medium plus DKN at the same dose+homogentisate at a    concentration of 5 mM.

It is very clear that, on medium 1, germination occurs normally, eachplantlet developing two cotyledons which are clearly green. Developmentthen takes place normally. On medium 2, germination occurs, but theplantlet which emerges is white, the two cotyledons exhibiting nopigmentation. The plantlets then die in a few days. On medium 3,germination occurs normally, the cotyledons are clearly green. Theplants develop, but very rapidly, the amount of homogentisate in themedium decreasing, the first symptoms of bleaching appear and plantgrowth stops, they end up dying as in the assay carried out on mediumNo. 2.

This makes it possible to confirm that HPPD is clearly the target forDKNs in plants and that it appears to be the only target. This alsoshows that homogentisate is transported from the culture medium to thecell site where it is necessary for correct functioning of the cell andsurvival of the plant.

Three strategies are currently available to make plants tolerant toherbicides, (1) detoxification of the herbicide with an enzyme whichconverts the herbicide, or its active metabolite, to nontoxicdegradation products, such as, for example, the enzymes for tolerance tobromoxynil or to basta (EP 242 236, EP 337 899); (2) mutation of thetarget enzyme to a functional enzyme less sensitive to the herbicide, orits active metabolite, such as, for example, the enzymes for toleranceto glyphosate (EP 293 356, S. R. Padgette et al., J. Biol. Chem., 266,33, 1991); or (3) overexpression of the sensitive enzyme, so as toproduce in the plant amounts of target enzyme which are sufficient withregard to the kinetic constants of this enzyme with respect to theherbicide in order to have sufficient functional enzyme, despite thepresence of its inhibitor.

This third strategy which has been described for successfully obtainingplants tolerant to HPPD inhibitors (WO 96/38567), it being understoodthat, for the first time, a strategy of simple overexpression of thesensitive (nonmutated) target enzyme, was used successfully to impart onplants tolerance at an agronomic level to a herbicide. Theidentification of HPPDs mutated in the C-terminal portion which exhibitimproved tolerance to HPPD inhibitors has made it possible to obtain animprovement in the tolerance of plants using the second strategy (WO99/24585).

The present invention consists of a novel method for making plantstolerant to a herbicide, which uses a novel or fourth strategy ofherbicide tolerance, this novel strategy comprising bypassing themetabolic pathway inhibited by said herbicide. This metabolic bypassingcan be summarized as follows:

-   for instance a herbicide “H” which is active by inhibiting the    activity of an enzyme “E” which converts the, substrate “S” to    product “P”, said product P and its metabolites being essential to    the life of the plant,-   the metabolic bypassing consists in expressing in the plant at least    one novel heterologous enzyme “NE” insensitive to “H” allowing    conversion of “S” to an intermediate product “I”, which product is    then converted into “P” either via the natural biosynthesis pathways    of the plant or via the expression of at least one other    heterologous enzyme “OE” also insensitive to “H”,-   the metabolic bypassing also consisting in expressing at least one    other heterologous enzyme “OE” insensitive to “H” allowing    conversion of “I” to “P”, “I” being an intermediate either naturally    produced by the plant or obtained by expressing at least one novel    heterologous enzyme “NE” insensitive to “H” allowing conversion of    “S” to “I”.

The present invention relates more particularly to a novel method formaking plants tolerant to HPPD inhibitors, said method comprising themetabolic bypassing of HPPD.

No pathway of metabolic bypassing has been described to date in plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic pathway of conversion of HPP to homogentisate.

FIG. 2 shows a schematic enzyme pathway starting with HPA.

FIG. 3 shows a diagram of a vector containing hppO and restrictionsites. FIG. 4 shows part of a 936 bp nucleotide sequence identified byPCR potentially encoding part of hppO (SEQ ID NO: 64) and complementarysequence (SEQ ID NO: 65)

FIG. 5 shows amino acid sequence alignments of HPPoxydase (upper SEQ IDNO: 66; lower SEQ ID NO: 72), D47069 (upper SEQ ID NO: 67; lower SEQ IDNO: 73), G69464 (upper SEQ ID NO: 68; lower SEQ ID NO: 74), YCEC1L(upper SEQ ID NO: 69; lower SEQ ID NO: 75), P37603 (upper SEQ ID NO: 70;lower SEQ ID NO: 76), and T34668 (upper SEQ ID NO: 71; lower SEQ ID NO:77).

FIG. 6 shows the construction of plasmid pBBR1MCS FT12Δ1.

FIG. 7 shows a diagram of the 5.2 kb insert of plasmid p5kbC.

FIG. 8 shows amino acid sequence alignments of HPAH, hydroxylase-Q53657,monoxygenase-Q9EAM4, putative oxygenase-Q5471, MHPA_ECOLI andOxygenase-086481 and consensus sequence.

In A, HPAH (SEQ ID NO: 78), hydroxylase-Q53657 (SEQ ID NO: 79),monoxygenase-Q9EAM4 (SEQ ID NO: 80), putative oxygenase-Q5471 (SEQ IDNO: 81), MHPA_ECOLI (SEQ ID NO: 82), Oxygenase-086481 (SEQ ID NO: 83)and consensus sequence (SEQ ID NO: 43).

In B, HPAH (SEQ ID NO: 84), hydroxylase-Q53657 (SEQ ID NO: 85),monoxygenase-Q9EAM4 (SEQ ID NO: 86), putative oxygenase-Q5471 (SEQ IDNO: 87), MHPA_ECOLI (SEQ ID NO: 88), Oxygenase-086481 (SEQ ID NO: 89).

In C, HPAH (SEQ ID NO: 90), hydroxylase-Q53657 (SEQ ID NO: 91),monoxygenase-Q9EAM4 (SEQ ID NO: 92), putative oxygenase-Q5471 (SEQ IDNO: 93), MHPA_ECOLI (SEQ ID NO: 94), Oxygenase-086481 (SEQ ID NO: 95)and consensus sequence (SEQ ID NO: 44).

In D, HPAH (SEQ ID NO: 96), hydroxylase-Q53657 (SEQ ID NO: 97),monoxygenase-Q9EAM4 (SEQ ID NO: 98), putative oxygenase-Q5471 (SEQ IDNO: 99), MHPA_ECOLI (SEQ ID NO: 100), Oxygenase-086481 (SEQ ID NO: 101)and consensus sequence (SEQ ID NO: 45).

FIG. 9 shows a diagram of plasmid pL1lac2.

FIG. 10 shows an expression cassette encoding an HPAC.

FIG. 11 shows an expression cassette encoding an HPAH.

FIG. 12 shows an expression cassette encoding HPP oxidase.

FIG. 13 shows an expression cassette encoding both HPAH and HPAC.

FIG. 14 shows an expression cassette encoding HPP oxidase, HPAH andHPAC.

It is known from the literature that the conversion of HPP tohomogentisate can be obtained by first carrying out conversion of HPP to4-hydroxyphenylacetic acid (4-HPA) with an enzyme extract exhibiting HPPoxidase activity, followed by conversion of 4-HPA to homogentisate withan enzyme extract exhibiting 4-HPA 1-hydrolase activity (WO 99/34008).This bypassing pathway is represented in FIG. 1.

A bibliographical study reveals that the enzyme activities required toconstruct the HPPD bypassing pathway were characterized on crudebacterial extracts in the 1970s. Thus, the HPP oxidase (HPPO, E.C.1.2.3.-) and 4-HPA 1-hydroxylase (HPAH, E.C. 1.14.13.18) activities wereidentified respectively in Arthrobacter globiformis (Blakley, 1977) andin Pseudomonas acidovdrans (Hareland et al., 1975). Since then, onlyHPAH has been purified, by Suemori et al., (1996), but neither theprotein sequence nor the nucleic acid, sequence are published. It istherefore necessary to identify the genes encoding these enzymeactivities.

In the bypassing pathway, the conversion of HPP to HGA takes place via4-HPA. Now, 4-HPA is a compound rarely identified in plants. It ispresent in Astilbe chinensis (Kindl, 1969), in Plantago sp. (Swiatek,1977) in dandelion (Taraxacum officinale; Dey & Harborne, 1997), inArtemisia (Swiatek et al., 1998), in the fruit of Forsythia suspensa(Liu et al., 1998) and, finally, in the marine alga Ulva lactuca (Flodinet al., 1999). There is little data regarding origin. It appears to beable to originate from tyrosine, from shikimate, or from tyramine. Thereis no more information on this regarding what becomes of it or its rolein plants. Kindl (1969) has shown its degradation via3,4-dihydroxyphenylacetic acid, while Flodin et al. (1999) havedemonstrated its conversion via 4-hydroxymandelic acid to2,4,6-tribromophenol, which accumulates in the green alga Ulva lactuca.Gross (1975) suggests that 4-HPA might be a growth regulator in certainhigher plants, and Abe et al. (1974) consider it to be an analog ofauxin in algae.

In order to implement the pathway of metabolic bypassing of HPPD, itwould have been necessary to identify and isolate beforehand the genesand the nucleic acid sequences encoding the enzyme(s) responsible forthe two activities above.

DEFINITIONS ACCORDING TO THE INVENTION

“Nucleic acid sequence”: a nucleotide or polynucleotide sequence, whichcan be of the DNA or RNA type, preferably of the DNA type, in particulardouble stranded. The nucleic acid sequence may be of natural origin, inparticular genomic DNA or cDNA, or else a synthetic or semisyntheticsequence, the nucleic acids comprising it having been chosen either tooptimize the codons of a coding sequence as a function of the hostorganism in which it will be expressed, or to introduce or eliminate oneor more restriction sites. Methods for preparing synthetic orsemisynthetic nucleic acid sequences are well known to those skilled inthe art.

“Sequence capable of selectively hybridizing”: the nucleic acidsequences which hybridize with a reference nucleic acid sequence at alevel significantly greater than the background noise. The backgroundnoise may be associated with the hybridization of other DNA sequencespresent, in particular of other cDNAs present in a cDNA library. Thelevel of the signal generated by the interaction between the sequencecapable of selectively hybridizing and the sequences defined by thesequence IDs above according to the invention is generally 10 times,preferably 100 times, greater than that of the interaction of the otherDNA sequences generating the background noise. The level of interactioncan be measured, for example, by labeling the probe with radioactiveelements such as ³²P. The selective hybridization is generally obtainedby using very severe conditions for the medium (for example 0.03 M NaCland 0.03 M sodium citrate at approximately 50° C.-60° C.). Thehybridization can of course be carried out according to the usualmethods of the state of the art (in particular Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual).

“Homolog of a nucleic acid sequence”: nucleic acid sequence exhibitingone or more sequence modifications compared to a reference nucleic acidsequence. These modifications can be obtained according to the usualtechniques of mutation, or else by choosing the syntheticoligonucleotides used in the preparation of said sequence byhybridization. With regard to the multiple combinations of nucleic acidswhich can result in the expression of the same amino acid, thedifferences between the reference sequence according to the inventionand the corresponding homolog may be considerable. Advantageously, thedegree of homology will be at least 60% compared to the referencesequence, preferably at least 70%, more preferentially at least 80%,even more preferentially at least 90%. These modifications are generallyand preferably neutral, that is to say that, for a coding sequence, theydo not affect the primary sequence of the protein or of the peptideencoded. They may, however, introduce nonsilent modifications, ormutations, which do not affect the function of the nucleic acid sequencecompared to the reference sequence. The methods for measuring andidentifying the homologies between nucleic acid sequences are well knownto those skilled in the art. The PILEUP or BLAST programs (in particularAltschul et al., 1993, J. Mol. Evol. 36:290-300; Altschul et al., 1990,J. Mol. Biol. 215:403-10) can for example be used.

“Fragments”: fragment of a reference nucleic acid or polypeptidesequence for which portions have been deleted but which conserves thefunction of said reference sequence.

“Heterolog”: nucleic acid sequence different from the nucleic acidsequence having the same function in a natural organism. A heterologoussequence may consist of a nucleic acid sequence modified in situ in itsnatural environment. It may also be a nucleic acid sequence isolatedfrom its natural organism then reintroduced into this same organism. Itmay also be a nucleic acid sequence which is heterologous with respectto another nucleic acid sequence, i.e. a sequence associated withanother sequence, this association not occurring naturally. This is inparticular the case of expression cassettes consisting of variousnucleic acid sequences which are not generally associated naturally.

“Homolog of a protein sequence”: protein sequences in which the primarysequence is different from the primary sequence of the referenceprotein, but which performs the same function as this referencesequence. The methods for measuring and identifying homologies betweenpolypeptides or proteins are also known to those skilled in the art. TheUWGCG package and the BESTFITT program can for example be used tocalculate the homologies (Deverexu et al., 1984, Nucleic Acid Res. 12,387-395).

“Expression cassette”: nucleic acid sequence comprising variousfunctional elements required for the expression of a coding sequence ina host organism. These functional elements comprise, in the direction oftranscription, a regulatory promoter sequence, also called promoter,functionally linked to a coding sequence and a regulatory terminatorsequence, also called terminator or stop. The expression cassette canalso comprise, between the regulatory promoter sequence and the codingsequence, regulatory elements such as transcription activators, orenhancers, and/or introns.

“Host organism”: according to the invention this is essentially intendedto mean plant cells or plants. For the cloning vectors, the hostorganisms can also be bacteria, fungi or yeasts.

“Plant cells”: cell which is derived from a plant and which canconstitute undifferentiated tissues such as calluses, differentiatedtissues such as embryos, parts of plants, plants or seeds.

“Plant”: differentiated multicellular organism capable ofphotosynthesis, in particular monocotyledonous or dicotyledonous, moreparticularly crop plants which may or may not be intended for animal orhuman food, such as rice, maize, wheat, barley, sugar cane, rapeseed,soybean, beetroot, potato, tobacco, cotton, clover, turf, or ornamentalplants such as petunias, or else banana plants, grapevines, raspberries,strawberries, tomatoes, salad plants, etc.

“Regulatory promoter sequence”: as a regulatory promoter sequence inplants, use may be made of any regulatory promoter sequence of a genewhich is naturally expressed in plants, in particular a promoter whichis expressed in particular in the leaves of plants, such as, forexample, “constitutive” promoters of bacterial, viral or plant origin,or else “light-dependent” promoters such as that of a gene of the plantribulose-biscarboxylase/oxygenase (RuBisCO) small subunit, or any knownsuitable promoter which can be used. Among the promoters of plantorigin, mention will be made of the histone promoters as described inapplication EP 0 507 698, or the rice actin promoter (U.S. Pat. No.5,641,876). Among the promoters of a plant virus gene, mention will bemade of that of the cauliflower mosaic virus (CAMV 19S or 35S), or ofCsVMV (U.S.), or the circovirus promoter (AU 689 311). Use may also bemade of a regulatory promoter sequence specific for particular regionsor tissues of plants, and more particularly seed-specific promoters([22] R. Datla et al., Biotechnology Ann. Rev. (1997) 3, 269-296),especially the promoters for napin (EP 255 378), for phaseolin, forglutenin, for heliantinin (WO 92/17580), for albumin (WO 98/45460), foroleosin (WO 98/45461), for ATS1 or for ATS3 (PCT/US98/06978, filed onOct. 20, 1998, incorporated herein by way of reference). Use may also bemade of an inducible promoter advantageously chosen from the, promotersfor phenylalanine ammonia lyase (PAL), for HMG-CoA reductase (HMG), forchitinases, for glucanases, for proteinase inhibitors (PI), for PRlfamily genes, for nopaline synthase (nos) or for the vspB gene (U.S.Pat. No. 5,670,349, Table 3), the. HMG2 promoter (U.S. Pat. No.5,670,349), the apple beta-galactosidase (ABG1) promoter or the appleaminocyclopropane carboxylate synthase (ACC synthase) promoter (WO98/45445).

“Transcription activators (enhancers)”: mention will be made, forexample, of the enhancer of the tobacco mosaic virus (TMV) described inapplication WO 87/07644, or of the tobacco etch virus (TEV) described byCarrington & Freed.

“Introns”: untranslated nucleic acid sequences. Mention will be made,for example, of intron 1 of the Arabidopsis histone gene as described inpatent application WO 97/04114 for expression in dicotyledonous plants,the rice actin first intron described in patent application WO 99/34005,or the maize adhl intron for expression in monocotyledonous plants.

“Coding sequence”: translated nucleic acid sequence. It comprises asequence encoding a protein or a peptide of interest, optionally fusedin the 5′ or in the 3′ position, with a sequence encoding a signalpeptide or a peptide for addressing to a particular cellularcompartment.

“Signal peptide or addressing peptide”: peptides fused to a protein or apeptide of interest in their N- or C-terminal portions, recognized bythe cellular machinery for addressing of the protein or of the peptideof interest to a particular cellular compartment. They are in particularchloroplast transit peptides for addressing the protein or the peptideof interest into chloroplasts, or signal peptides to various cellularcompartments, for example the vacuole, the mitochondria, the endoplasticreticulum, the golgi apparatus, etc. The role of such protein sequencesis in particular described in issue 38 of the review Plant molecularBiology (1998) devoted in large part to the transport of proteins intothe various compartments of the plant cell (Sorting of proteins tovacuoles in plant cells pp 127-144; the nuclear pore complex pp 145-162;protein translocation into and across the chloroplastic envelopemembranes pp 91-207; multiple pathways for the targeting of thylakoidproteins in chloroplasts pp 209-221; mitochondrial protein import inplants pp 311-338).

“Chloroplast transit peptide”: the chloroplast transit peptide isencoded by a: nucleic acid sequence positioned 5′ of the nucleic acidsequence encoding a protein or a peptide of interest, so as to allow theexpression of a transit peptide/protein (peptide) of interest fusionprotein. The transit peptide makes it possible to address the protein orthe peptide of interest into plasts, more particularly chloroplasts, thefusion protein being cleaved between the transit peptides and theprotein or the peptide of interest as it passes through the plastmembrane. The transit peptide may be single, such as an EPSPS transitpeptide (U.S. Pat. No. 5,188,642) or a transit peptide from theribulose-biscarboxylase/oxygenase small subunit (RuBisCO ssu) of aplant, optionally comprising some amino acids of the N-terminal portionof the mature RuBisCO ssu (EP 189 707), or else a multiple transitpeptide comprising a first plant transit peptide fused to a portion ofthe N-terminal sequence of a mature protein located in plastids, fusedto a second plant transit peptide as described in patent EP 508 909, andmore particularly the optimized transit peptide comprising a transitpeptide of sunflower RuBisCO ssu fused to 22 amino acids of theN-terminal end of maize RuBisCO ssu fused to the transit peptide ofmaize RuBisCO ssu as described with its coding sequence in patent EP 508909.

“Signal peptide”: these peptide sequences are in particular described inissue 38 of the review Plant molecular Biology (1998) devoted in largepart to the transport of proteins into the various compartments of theplant cell (Sorting of proteins to vacuoles in plant cells pp 127-144;the nuclear pore complex pp 145-162; protein translocation into andacross the chloroplastic envelope membranes pp 91-207; multiple pathwaysfor the targeting of thylakoid proteins in chloroplasts pp 209-221;mitochondrial protein import in plants pp 311-338). Peptides foraddressing to the vacuole are widely described in the literature (J. M.Neuhaus and J. C. Rogers Sorting of proteins to vacuoles in plant cellsPlant molecular Biology 38: 127-144, 1998). Preferably, the vacuolepeptide is the vacuole peptide of the protein described in J. M. Ferulloet al. (Plant Molecular Biology 33: 625-633, 1997), fused to theC-terminal portion of the protein or of the peptide of interest.

“Regulatory terminator sequence”: also comprising the polyadenylationsequences, this is intended to mean any sequence which is functional inplant cells or plants, whether of bacterial origin, such as, forexample, the nos terminator of Agrobacterium tumefaciens, of viralorigin, such as, for example, the CaMV 35S terminator, or else of plantorigin, such as, for example, a histone terminator as described inapplication EP 0 633 317.

“Vector”: cloning and/or expression vector for the transformation of ahost organism, containing at least one expression cassette. The vectorcomprises, beside the expression cassette, at least one origin ofreplication. The vector may consist of a plasmid, a cosmid, abacteriophage or a virus, transformed by introducing the expressioncassette. Such transformation vectors, as a function of the hostorganism to be transformed, are well known to those skilled in the artand widely described in the literature. For the transformation of plantcells or plants, it will in particular be a virus which can be used totransform developed plants and which also contains its own elements forreplication and for expression.

Preferentially, the vector for transforming plant cells or plants is aplasmid.

HPP Oxidase

A first subject of the invention concerns a nucleic acid sequenceencoding an HPP oxidase, and the corresponding polypeptide.Preferentially, the HPP oxidase is insensitive to HPPD inhibitors, inparticular to isoxazoles such as isoxaflutole and their diketonitriles,in particular those defined above. The HPP oxidase is in particular anHPP oxidase of bacterial origin, for example from Arthrobacter, inparticular from Arthrobacter globiformis. The HPP oxidase isadvantageously a protein the primary amino acid sequence of which isrepresented by sequence identifier No. 2 (SEQ ID No. 2) the sequenceshomologous thereto and the fragments thereof.

Protein sequences of HPP oxidases homologous to SEQ ID No. 2 are inparticular represented by SEQ ID Nos. 4 and 6, the sequences homologousthereto and the fragments thereof.

The HPP oxidase represented by SEQ ID No. 4 corresponds to the HPPoxidase of SEQ ID No. 2 for which a glycine is replaced with an alanine.

The present invention also relates to a nucleic acid sequence encodingan HPP oxidase as defined above.

Preferentially, the sequence encoding the HPP oxidase is a DNA sequence,especially genomic DNA or cDNA, in particular a heterologous or isolatedsequence.

The sequence encoding an HPP oxidase according to the invention is inparticular chosen from, the coding sequences of the DNA sequencesrepresented by SEQ ID No. 1, 3, 5 or 15, the sequences homologousthereto, the fragments thereof, and the sequences capable of selectivelyhybridizing to SEQ ID 1, 3, 5 or 15.

The coding sequence of SEQ ID No. 5 comprises three mutations atpositions 463, 602 and 1511 relative to SEQ ID No. 1, which are silent,i.e. which introduce no modification of the corresponding polypeptide.

4-HPA 1-hydroxylase

A second subject of the invention concerns the means required for theexpression of 4-HPA 1-hydroxylase. Contrary to what was expected fromthe literature regarding the activity of certain protein extracts, itwas noted that the 4-HPA 1-hydroxylase activity in the bacteria, inparticular Pseudomonas, resulted from the sum of the activity of twoenzymes, hereinafter referred to HPAH and HPAC.

HPAH

HPAH allows the conversion of HPA to an intermediate metabolite,hereinafter referred to as metabolite Z, the structure of which remainsundetermined. It may be seriously envisaged that HPAH allowshydroxylation of the aromatic ring of HPA, the metabolite Z stabilizingin the form of a ketone. This hypothesis of enzyme activity isrepresented in FIG. 2.

A second subject of the invention therefore concerns a nucleic acidsequence encoding an HPAH, and the corresponding polypeptide.Preferentially, the HPAH is insensitive to HPPD inhibitors, inparticular to isoxazoles such as isoxaflutole and their diketonitriles,especially those defined above. The HPAH is in particular an HPAH ofbacterial origin, for example from Pseudomonas, in particular fromPseudomonas acidovorans. The HPAH is advantageously a protein theprimary amino acid sequence of which is represented by sequenceidentifiers No. 8 and 18 (SEQ ID No. 8 and SEQ ID No. 18), the sequenceshomologous thereto and the fragments thereof.

The present invention also relates to a nucleic acid sequence encodingan HPAH as defined above.

Preferentially, the sequence encoding the HPAH is a DNA sequence,especially genomic DNA or cDNA, in particular a heterologous or isolatedsequence.

The sequence encoding an HPAH according to the invention is inparticular chosen from the coding regions of the sequences representedby SEQ ID No. 7 or 17 the sequences homologous thereto, the fragmentsthereof, and the sequences capable of selectively hybridizing to SEQ IDNo. 7 or 17.

HPAC

HPAC is the second enzyme which allows conversion of the metabolite Z tohomogentisate.

A third subject of the invention therefore concerns a nucleic acidsequence encoding an HPAC, and the corresponding polypeptide.Preferentially, the HPAC is insensitive to HPPD inhibitors, inparticular to isoxazoles such as isoxaflutole and their diketonitriles,especially those defined above. The HPAH is in particular an HPAC ofbacterial origin, for example from Pseudomonas, in particular fromPseudomonas acidovorans. The HPAC is advantageously a protein theprimary amino acid sequence of which is represented by sequenceidentifier No. 10 (SEQ ID No. 10), the sequences homologous thereto andthe fragments thereof.

Protein sequences of HPAC homologous to SEQ ID No. 10 are in particularrepresented by SEQ ID Nos. 12, 14 and 20, the sequences homologousthereto and the fragments thereof.

The present invention also relates to a nucleic acid sequence encodingan HPAC as defined above.

Preferentially, the sequence encoding the HPAC is a DNA sequence,especially genomic DNA or cDNA, in particular a heterologous or isolatedsequence.

The sequence encoding an HPAC according to the invention is inparticular chosen from the coding regions of the sequences representedby SEQ ID No. 9, 11, 13 or 19, the sequences homologous thereto, thefragments thereof, and the sequences capable of selectively hybridizingto SEQ ID No. 9, 11, 13 or 19.

Expression Cassettes

The present invention also relates to an expression cassette the codingsequence of which comprises a nucleic acid sequence selected from thenucleic acid sequences encoding an HPP oxidase, an HPAH or HPAC asdefined above.

The coding sequence can also comprise, in the 5′ or in the 3′ position,a sequence encoding a signal peptide or a transit peptide.Advantageously, the coding sequence comprises, positioned 5′ of thesequence encoding HPP oxidase, an HPAH or an HPAC, and a sequenceencoding a transit peptide for chloroplast addressing, in particular amultiple transit peptide, more particularly the optimized transitpeptide.

The present invention therefore also relates to a transit peptide/HPPoxidase, transit peptide/HPAH or transit peptide/HPAC fusion protein,the sequence of the transit peptide being defined above, in particularthe sequence of the optimized transit peptide as described in patentapplication EP 508 909.

Preferentially, the regulatory promoter sequence is chosen from theregulatory promoter sequences allowing constitutive expression of thecoding sequence. These are in particular the sequences of the CaMV 35S,CsVMV, rice actin or histone promoters.

It is also possible to choose to express the coding sequences accordingto the invention at a level of expression close to the level ofexpression of the gene intended to be bypassed. Use may be made, in theexpression cassette according to the invention, of a regulatory promotersequence chosen from the regulatory promoter sequences of plant HPPDs.

For expression of the three enzymes HPP oxidase, HPAH and HPAC in thesame plant, it is possible choose the expression cassettes for thecorresponding coding sequences, different regulatory promoter sequencesexhibiting different expression profiles, by virtue of their strengthand/or their location in the various functional organs of the plant.

Regulatory promoter sequences allowing a gradient of expressionHPAC>HPAH>HPP oxidase, or vice versa, may be chosen.

For expression of HPP oxidase, of the HPAH and of the HPAC, theregulatory promoter sequence is advantageously chosen from the groupcomprising the promoters of plant HPPD, of histone H3 or H4, especiallyfrom Arabidopsis or from maize, in particular those described in patentapplication EP 507 698, and of plant RuBisCO SSU, in particular fromsunflower or from maize as described in patent application WO 99/25842,the CaMV 35S promoter or the CsVMV promoter, and combinations thereof,in particular the histone/35S hybrid promoters as described in theexamples of patent application EP 507 698. For expression inmonocotyledonous plants, these regulatory promoter sequences willadvantageously be combined with the first intron of rice actin.

According to one embodiment of the invention, the expression cassetteencoding an HPP oxidase comprises a histone promoter, a sequenceencoding an HPP oxidase and a histone terminator (FIG. 12; SEQ ID No.15).

According to another embodiment of the invention, the expressioncassette encoding an HPAH comprises a CaMV 35S promoter, a sequenceencoding an HPAH and a NOS terminator (FIG. 11; SEQ ID No. 17).

According to another embodiment of the invention, the expressioncassette encoding an HPAC comprises a CsVMV promoter, a sequenceencoding an HPAC and a NOS terminator (FIG. 10; SEQ ID No. 19).

Vectors:

The present invention also relates to a cloning and/or expression vectorcomprising at least one expression cassette according to the invention.

According to a first embodiment of the invention, the vector comprisesjust one of the expression cassettes according to the invention, chosenfrom the cassettes comprising a coding sequence for an HPP oxidase, anHPAH or an HPAC as defined above.

According to a second embodiment of the invention, the vector comprisestwo expression cassettes according to the invention, chosen from thecassettes comprising a coding sequence for an HPP oxidase, an HPAH or anHPAC as defined above, combined in pairs in the same vector: HPP oxidaseand HPAH, HPP oxidase and HPAC, HPAH and HPAC.

A vector comprising an expression cassette encoding the HPAH and anotherencoding the HPAC can comprise the combination of the two expressioncassettes defined above (SEQ ID No. 17 and 19). Such an expressioncassette is represented in FIG. 13 and SEQ ID No. 21.

According to a third embodiment of the invention, the vector comprisesthree expression cassettes according to the invention, a firstexpression cassette for the HPP oxidase, a second expression cassettefor the HPAH and a third expression cassette for the HPAC. Such anexpression cassette can comprise, the combination of the three cassettesdefined above (SEQ ID Nos. 15, 17 and 19). Such a vector is representedin FIG. 14 and SEQ ID No. 22.

The vectors according to the invention as defined above can alsocomprise expression cassettes for other proteins or peptides ofinterest.

When the vector comprises several expression cassettes, these cassettescan have various orientations in pairs with respect to one another,colinear, divergent or convergent.

The expression cassettes for other proteins or peptides of interestcomprise a nucleic acid sequence encoding proteins or peptides ofinterest different than the HPP oxidase, the HPAH and the HPAC definedabove.

There may be sequences of a gene encoding a selectable marker, such as agene imparting novel agronomic properties to the transformed plant, or agene which improves the agronomic quality of the transformed plant.

Selectable Markers

Among the genes encoding selectable markers, mention may be made of thegenes for resistance to antibiotics, the genes for tolerance toherbicides, (bialaphos, glyphosate or isoxazoles), genes encodingreadily identifiable reporter enzymes such as the GUS enzyme, genesencoding pigments or enzymes regulating the production of pigments inthe transformed cells. Such selectable marker genes are in particulardescribed in patent applications EP 242 236, EP 242 246, GB 2 197 653,WO 91/02071, WO 95/06128, WO 96/38567 or WO 97/04103.

Genes of Interest

Among the genes which impart novel agronomic properties to thetransformed plants, mention may be made of the genes which imparttolerance to certain herbicides, those which impart resistance tocertain insects, those which impart tolerance to certain diseases, etc.Such genes are in particular described in patent applications WO91/02071 and WO 95/06128.

Herbicide Tolerance

The present invention is particularly suitable for the expression ofgenes which impart tolerance to certain herbicides to the transformedmonocotyledonous plants and plant cells. Among the genes impartingtolerance to certain herbicides, mention may be made of the Bar geneimparting tolerance to bialaphos, the gene encoding a suitable EPSPSimparting resistance to herbicides having EPSPS as the target, such asglyphosate and its salts (U.S. Pat. No. 4,535,060, U.S. Pat. No.4,769,061, U.S. Pat. No. 5,094,945, U.S. Pat. No. 4,940,835, U.S. Pat.No. 5,188,642, U.S. Pat. No. 4,971,908, U.S. Pat. No. 5,145,783, U.S.Pat. No. 5,310,667, U.S. Pat. No. 5,312,910, U.S. Pat. No. 5,627,061,U.S. Pat. No. 5,633,435, FR 2 736 926), the gene encoding glyphosateoxidoreductase (U.S. Pat. No. 5,463,175), or else a gene encoding anHPPD imparting tolerance to herbicides having HPPD as the target, suchas isoxazoles, in particular isoxafutole (FR 95 06800, FR 95 13570),diketonitriles (EP 496 630, EP 496 631) or triketones, in particularsulcotrione (EP 625 505, EP 625 508, U.S. Pat. No. 5,506,195). Suchgenes encoding an HPPD imparting tolerance to herbicides having HPPD asthe target are described in patent application WO 96/38567.

Among the genes encoding a suitable EPSPS imparting resistance toherbicides having EPSPS as the target, mention will be made moreparticularly of the gene encoding a plant EPSPS, in particular frommaize, exhibiting two mutations, 102 and 106, described in patentapplication FR 2 736 926, referred to below as EPSPS double mutant, orelse the gene encoding an EPSPS isolated from Agrobacterium described bysequence ID 2 and ID 3 of U.S. Pat. No. 5,633,435, referred to below asCP4.

Among the genes encoding an HPPD imparting tolerance to herbicideshaving HPPD as the target, mention will be made more particularly of theHPPD from Pseudomonas and that from Arabidopsis, described in patentapplication WO 96/38567.

In the cases of the genes encoding EPSPS or HPPD, and more particularlyencoding the genes above, the sequence encoding these enzymes isadvantageously preceded by a sequence encoding a transit peptide, inparticular encoding the transit peptide termed “optimized transitpeptide” described in U.S. Pat. No. 5,510,471 or 5,633,448.

Resistance to Insects

Among the proteins of interest imparting novel properties of resistanceto insects, mention will be made more particularly of the Bt proteinswidely described in the literature and well known to those skilled inthe art. Mention will also be made of the proteins extracted frombacteria such as Photorabdus (WO 97/17432 and WO 98/08932).

Resistance to Diseases

Among the proteins or peptides of interest imparting novel properties ofresistance to diseases, mention will be made in particular ofchitinases, glucanases and oxalate oxidase, all these proteins and theircoding sequences being widely described in the literature or elseantibacterial and/or antifungal peptides, in particular cysteine-richpeptides of less than 100 amino acids, such as plant thionins ordefensins, and more particularly lytic peptides of all originscomprising one or more disulfide bridges between the cysteines andregions comprising basic amino acids, in particular the following lyticpeptides: androctonin (WO 97/30082 and PCT/FR98/01814, filed on Aug. 18,1998) or drosomycin (PCT/FR98/01462, filed on Jul. 8, 1998).

According to a particular embodiment of the invention, the protein orpeptide of interest is chosen from fungal elicital peptides, inparticular elicitins (Kamoun et al., 1993; Panabières et al., 1995).

Modification of the Quality

Mention may also be made of the genes which modify the constitution ofthe modified plants, in particular the content and the quality ofcertain essential fatty acids (EP 666 918) or else the content and thequality of the proteins, in particular in the leaves and/or the seeds ofsaid plants. Mention will in particular be made of the genes encodingproteins enriched in sulfur-containing amino acids (A. A. Korit et al.,Eur. J. Biochem. (1991) 195, 329-334; WO 98/20133; WO 97/41239; WO95/31554; WO 94/20828; WO 92/14822). These proteins enriched insulfur-containing amino acids will also have the function of trappingand storing excess cysteine and/or methionine, making it possible toavoid possible problems of toxicity associated with overproduction ofthese sulfur-containing amino acids by trapping them. Mention may alsobe made of genes encoding peptides rich in sulfur-containing aminoacids, and more particularly in cysteines, said peptides also having anantibacterial and/or antifungal activity. Mention will more particularlybe made of plant defensins, along with lytic peptides of any origin, andmore particularly the following lytic peptides: androctonin (WO 97/30082and PCT/FR98/01814, filed on Aug. 18, 1998) or drosomycin(PCT/FR98/01462, filed on Jul. 8, 1998).

Plant Cells and Transgenic Plants

The present invention also relates to transformed plant cells and plantscomprising at least one expression cassette for an HPP oxidase, for anHPAH or for an HPAC as defined above.

According to a first embodiment of the invention, the plant cells or theplants comprise just one of the expression cassettes according to theinvention, chosen from the cassettes comprising a coding sequence for anHPP oxidase, an HPAH or an HPAC as defined above.

According to a second embodiment of the invention, the plant cells orthe plants comprise two expression cassettes according to the invention,chosen from the cassettes comprising a coding sequence for an HPPoxidase, an HPAH or an HPAC as defined above, combined in pairs in thesame vector: HPP oxidase and HPAH, HPP oxidase and HPAC, HPAH and HPAC.

According to a third embodiment of the invention, the plant cells or theplants comprise three expression cassettes according to the invention, afirst expression cassette for HPP oxidase, a second expression cassettefor HPAH and a third expression cassette for HPAC.

The plant cells or the plants according to the invention as definedabove can also comprise expression cassettes for other proteins orpeptides of interest defined above.

Preferentially, the expression cassettes are stably integrated into thegenome of the plant cells or of the plants. More preferentially, theplants according to the invention are fertile, the expression cassettesaccording to the invention being transferred to their descendance.

The present invention also relates to seeds of transgenic plants above,which seeds comprise an expression cassette according to the inventionencoding an HPP oxidase, an HPAH or an HPAC.

The various expression cassettes in the transformed plants according tothe invention can originate either from the same transformed parentplant, and in this case, the plant is derived from a single process oftransformation/regeneration with the various expression cassettescontained in the same vector or by cotransformation using severalvectors. It may also be obtained by crossing parent plants eachcontaining at least one expression cassette according to the invention.

Transformation of the Plant Cells and of the Plants

A subject of the invention is also a method of transforming the plantcells and the plants by introducing at least one nucleic acid sequenceor an expression cassette according to the invention as defined above,which transformation can be obtained by any suitable known means, widelydescribed in the specialized literature and in particular the referencescited in the present application, more particularly with the vectoraccording to the invention.

A series of methods consists in bombarding cells, protoplasts or tissueswith particles to which the DNA sequences are attached. Another seriesof methods consists in using, as means for transfer into the plant, achimeric gene inserted into an Agrobacterium tumefaciens Ti plasmid oran Agrobacterium rhizogenes Ri plasmid. Other methods can be used, suchas microinjection or electroporation, or else direct precipitation usingPEG. Those skilled in the art will choose the appropriate method as afunction of the nature of the host organism, in particular of the plantcell or of the plant.

When the desire is to introduce several nucleic acid sequences orexpression cassettes, it can be done using a single vector according tothe invention comprising the various expression cassettes. They may alsobe introduced into the host organism by cotransformation using severalvectors, each one comprising at least one expression cassette.

In general, the transgenic plants according to the invention areobtained by transformation of plant cells and then regeneration of aplant, preferably fertile, from the transformed cell. The regenerationis obtained by any suitable method, which depends on the nature of thespecies, such as for example described in the references above. For themethods of transforming the plant cells and of regenerating the plants,mention will in particular be made of the following patents and patentapplications: U.S. Pat. No. 4,459,355, U.S. Pat. No. 4,536,475, U.S.Pat. No. 5,464,763, U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,187,073, EP267,159, EP 604 662, EP 672 752, U.S. Pat. No. 4,945,050, U.S. Pat. No.5,036,006, U.S. Pat. No. 5,100,792, U.S. Pat. No. 5,371,014, U.S. Pat.No. 5,478,744, U.S. Pat. No. 5,179,022, U.S. Pat. No. 5,565,346, U.S.Pat. No. 5,484,956, U.S. Pat. No. 5,508,468, U.S. Pat. No. 5,538,877,U.S. Pat. No. 5,554,798, U.S. Pat. No. 5,489,520, U.S. Pat. No.5,510,318, U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,405,765, EP 442 174,EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO95/06128.

Selective Weeding

A subject of the invention is also a method for selective weeding ofplants, in particular crops, using an HPPD inhibitor, in particular aherbicide defined above, characterized in that this herbicide is appliedto transformed plants according to the invention, equally in pre-sowing,in pre-emergence and in post-emergence of the crop.

The present invention also relates to a method of weed killing in anarea of a field comprising seeds or transformed plants according to theinvention, which method comprises the application, in said area of thefield, of a dose, which is toxic for said weeds, of an HPPD-inhibitingherbicide, without however substantially affecting the seeds ortransformed plants according to the invention.

The present invention also relates to a method of growing thetransformed plants according to the invention, which method comprisessowing the seeds of said transformed plants in an area of a fieldsuitable for growing said plants, applying to said area of said field adose, which is toxic for the weeds, of a herbicide having HPPD as thetarget, defined above, in the event of weeds being present, withoutsubstantially affecting said seeds or said transformed plants, thenharvesting the plants grown, when they have reached the desired maturityand, optionally, separating the seeds from the harvested plants.

According to the invention, the expression “without substantiallyaffecting said seeds or said transformed plants” is intended to meanthat the transformed plants according to the invention, subjected toapplication of a dose of herbicide which is toxic for the weeds, exhibitslight or zero phytotoxicity. According to the invention, the expression“dose which is toxic for the weeds” is intended to mean an applied doseof the herbicide for which the weeds are killed. According to theinvention, the, term “slight phytotoxicity” is intended to mean apercentage of bleached leaves of less than 25%, preferentially less than10%, more preferentially less than 5%. It is also understood accordingto the present invention that application of the same toxic dose to aplant which is otherwise comparable but not transformed, i.e. which doesnot comprise at least one expression cassette according to theinvention, would lead to the observation on said plant of phytoxicitysymptoms greater than those observed for the transformed plant accordingto the invention.

In the two methods above, the application of the herbicide having HPPDas the target can be carried out according to the invention, equally inpre-sowing, in pre-emergence and in post-emergence of the crop.

For the purpose of the present invention, the term “herbicide” isintended to mean a herbicidal active material alone or combined with anadditive which modifies its effectiveness, such as, for example, anagent which increases the activity (synergist) or which limits theactivity (safener). The HPPD-inhibiting herbicides are in particulardefined previously. Of course, for their practical application, theherbicides above are combined, in a manner known per se, with theadjuvants of formulations conventionally used in agrochemistry.

When the transformed plant according to the invention comprises anothergene for tolerance to another herbicide (such as, for example, a geneencoding an EPSPS, which may or may not be mutated, imparting to theplant tolerance to glyphosate), or when the transformed plant isnaturally insensitive to another herbicide, the method according to theinvention may comprise the simultaneous application or the applicationat a different time of an HPPD inhibitor in combination with saidherbicide, for example glyphosate.

The various aspects of the invention will be understood more clearlythrough the experimental examples below.

All the methods or operations described below in these examples aregiven by way of examples and correspond to a choice, made from thevarious methods available to achieve the same result. This choice has nobearing on the quality of the result and, consequently, any suitablemethod can be used by those skilled in the art to achieve the sameresult. Most of the methods for engineering DNA fragments are describedin Coligan et al., (1995), Ausubel et al., (1995); Maniatis et al.,(1982) and Sambrook et al.

The bibliographical references cited above are integrated into thepresent patent application by way of reference, in particular thebibliographical references defining the nucleic acid sequences encodingnative, chimeric or mutated HPPDs, optionally combined with a signalpeptide or a transit peptide.

EXAMPLE I Identification of the Gene Encoding the HPP Oxidase fromArtbrobacter Globiformis

HPP oxidase (HPPO) converts HPP to 4-HPA via a decarboxylation reaction.This enzyme therefore catalyzes the first enzyme activity required toconstruct the metabolic pathway bypassing HPPD. HPP oxidase activity hasbeen characterized in crude extracts of Rhodococcus erythropolis S1(Suemori et al., 1995) or in a partially purified extract ofArthrobacter globiformis (Blakley, 1977). To our knowledge, the proteinhas not been purified. In order to be able to introduce this enzymeactivity into the plant, it is necessary to identify the gene thereof.Various approaches can be envisaged: (1) insertional mutagenesis andtherefore identification of the gene through the loss of the enzymeactivity, (2) functional complementation of a microorganism using agenomic library, (3) purification of the protein in order to work backto the nucleic acid sequence.

The three approaches were used. The functional complementation and theinsertional mutagenesis will be developed relatively little, since thesetechniques do not make it possible to identify the HPPO gene.

I.1 Materials and Methods

I.1.1—Culturing Conditions

I.1.1.1—Rich Media

Luria-Bertani (LB; sold by Bio101) medium is used to culture thebacteria (E. coli, P. fluorescens) in the molecular biology experiments.For culturing A. globiformis Columbia-ANC medium enriched with 5% ofsheep blood (BioMèrieux) will be preferred. This rich medium containstwo antibiotics (nalidixic acid and colimycin) which inhibitGram-negative microorganisms. Although the three bacteria grow on richmedium at 37° C., A. globiformis and P. fluorescens are generallycultured at 29° C.

I1.1.2—M^(Ag) Culture Medium

The culture medium described by Blakley (1977) precipitates, and it istherefore necessary to filter it before use. We gradually changed themedium in order to achieve an optimal “minimal” medium. The factorsconsidered are the growth rate of A. globiformis and the enzyme activityof HPPO. The medium selected (M^(Ag)) is an M9 medium (Maniatis et al.,1982) which is slightly modified: Na₂HPO₄, 12H₂O (6 g/L); KH₂PO₄ (3g/L); NH₄Cl (1 g/L); NaCl (0.5 g/L); CaCl₂ (6 mg/L); FeSO₄ 7H₂O (6mg/L); yeast extract (20 mg/L); and, finally, the substrate (HPP ortyrosine or citrate) at a concentration of 1 g/L. The medium isautoclaved. Before use, 1 mL of sterile 1 M MgSO₄ is added per liter ofmedium.

This minimum medium is also used to culture P. fluorescens

I.1.2—Construction of an Arthzrobacter globiformis Genomic Library

There is no reliable technique for making a complete bacterial cDNAlibrary. We therefore decided to create an Arthrobacter globiformisgenomic library. To produce this, we chose the cosmid system. The cosmidlibrary was prepared for the functional complementation experiments andwas then used later to search for the cosmid(s) containing the hppOgene.

I.1.2.1—The Cosmid Vector pLAFR5

I.1.2.1.1—Description of the Vector

We choose the conjugated cosmid vector pLAFR-5 (Keen et al., 1988) whichcan accept an insert of approximately 20 kb. Equipped with an origin oftransfer and an origin of replication with a broad Gram-negative-hostspectrum, it can be transmitted to other bacterial genera bytri-parenteral conjugation, which can be useful for testing functionalcomplementation in various bacterial genera. It imparts resistance totetracycline.

I.1.2.1.2—Preparation of the Vector

The plasmid pLAFR-5 is purified using an alkaline lysis protocol(Maniatis et al., 1982), treated with RNAse and then digested with BamHI and Sca I. The digestion with Bam HI makes it possible to open thesite into which the inserts of genomic DNA digested with Sau3A will be“ligated”. The digestion with Sca I makes it possible to release the cossites which allow the encapsidation. After extraction with phenol thenchloroform, the DNA is precipitated with ethanol. The dry DNA isdissolved in water. The vector thus prepared is stored at −20° C.

I.1.2.1—Preparation of the A. globiformis Genomic DNA

A 24-hour culture (200 mL, 180 rpm, 29° C.) prepared in the medium (200mL) described by Blakley (1977) is centrifuged at 3000 g at 4° C. for 15minutes. The cell pellet, taken up with 10 mL of lysis solution (TE pH8; 0.5% SDS; 1 mg proteinase K), is incubated at 37° C. in a waterbathwith gentle agitation every 20 minutes. After 90 minutes, the suspensionof lysed cells is poured into a polypropylene JA-20 tube. 10 mL ofphenol/chloroform/isoamyl alcohol (25/24/1) are then added, followed bycentrifugation at 6000 g for 15 minutes at 4° C. The supematant is thentransferred into a new JA20 tube, to which 1.8 mL of 10 M ammoniumacetate and 10 mL of isopropanol are added. After centrifugation at 20000 g for 20 minutes at 4° C., the pellet is rinsed with 70% ethanol.The dry pellet is taken up with 1 mL of TE, pH 8, and then transferredinto a 2 mL Eppendorf tube to which 10 Lii of RNAse (10 mg.mL⁻¹) areadded. After 30 min at 37° C., 800 μL of phenol/chloroform/isoamylalcohol are added. After centrifugation, the supernatant is transferredto a new Eppendorf tube and extracted with 0.8 mL of chloroform. Thesupematant is then transferred into a final Eppendorf tube, to which 200μL of 10 M ammonium acetate and 800 μL of isopropanol are added. Aftercentrifugation, the pellet is rinsed with 70% ethanol and then, oncedry, taken up in 500 μL of water. The genomic DNA is then stored at −20°C.

I.1.2.3—Controlled Digestion of the A. globiformis Genomic DNA

Only cosmids of 40-45 kb can be encapsidated. Since the vector is 21.5kb, the inserts of A. globiformis genomic DNA should be between 19 and22 kb in size. These fragments are obtained by performing a controlleddigestion of the Arthrobacter globiformis genomic DNA. In order todefine the optimal conditions for the controlled digestion, digestion ofthe A. globiformis genomic DNA are carried out with varying amounts ofSau 3A restriction enzyme. It, appears that the best digestion conditionuses 0.08, Sau 3A enzyme units for 30 minutes at 37° C. The genomic DNAthus digested is between 15 and 22 kb in size. The genomic DNA thusdigested is extracted with phenol, then with chloroform and, finally,precipitated with ethanol.

I.1.2.4 —Ligation of A. globiormis Genomic DNA into the Cosmid Vector

The ligation reaction is carried out in a final volume of 10 μLcontaining 500 ng of pLAFR-5 digested with Bam HI and Sca I, 650 ng ofgenomic DNA digested with Sau 3A, 320 units of T₄ DNA ligase (N. E. B. )and 5 mM of ATP. The ligation takes place at 12° C. overnight(approximately 16 hours). The 5 mM of ATP makes it possible to avoidligations between blunt ends (Sca I) (Feretti & Sgaramella, 1981) suchthat the dimers of vectors having no insert cannot become encapsidatedin the head of the λ phages.

I.1.2.5—Encapsidation of the Cosmids and Amplification of the CosmidLibrary

The encapsidation of the cosmids, carried out using the GIGAPACK II XLkit (Stratagene) respecting the supplier's instructions, provides anefficiency of transfection greater than those obtained with conventionaltransformation techniques. To amplify the cosmid library, Keen et al.(1988) advise using Escherichia coli DH-1 and HB101. Specifically, whenthe strains are cultured on maltose, they produce a membrane-boundprotein which enables better attachment of the phage and therefore moreefficient transfection of the cosmids. The library, amplified accordingto Stratagene's recommendations, is stored at −80° C. To evaluate thecosmid library, the plasmid DNA isolated from about thirty clones isdigested with Apa I or Eco RI. The restriction profiles are reserved ona 0.8% agarose gel.

I.1.3—Purification of the HPP Oxidase

I.1.3.1—Colorimetric Assay for the HPP Oxidase Activity

In order to be able to control the purification steps, the HPP oxidaseactivity is followed using the colorimetric assay described by Blakley(1977). The enzyme reaction is stopped by adding2,4-dinitrophenylhydrazine (2,4-DNPH), in solution in 2 M HCl-Id. The2,4-DNPH reacts with the ketone function in the alpha position of acarboxylic function (example: HPP). A hydrazone thus forms, which can berevealed by basifying the medium. When the HPP is completely convertedto 4-HPA during the enzyme reaction, the hydrazone cannot form, and thecharacteristic yellow color of 2,4-DHPA is therefore obtained in basicmedium. If the HPP is not completely converted to 4-HPA during theenzyme reaction, the formation of hydrazone is possible. Thesehydrazones are brown in color in basic medium. A variation in colorbetween these two extremes is obtained as a function of the amount ofHPP consumed. The absorption measurements are carried out at 445 or 450nm. In order to make this assay more easy to handle, we adapted it tothe 96-well microplate format. The reaction mixture comprises GSH (900μM); HPP (135 μM); TPP (1.8 mM); MgCl₂ (4.5 mM); FAD (4 μM); potassiumphosphate buffer (90 mM) pH 7.4. The mixture is kept on ice. 50 μl ofthe test fraction and 150 μL of reaction mixture are placed in eachwell. After 20 min at 30° C., the enzyme reaction is stopped with 13 μLof 2,4-DNPH solution (0.1% in 2 M HCl). The mixture is left to react for20 min at ambient temperature. The formation of hydrazone is revealed byadding 13 μL of 10 M NaOH solution. To prepare the standard range,reaction mixtures with varying concentrations of HPP are prepared. The50 μL protein fractions are replaced with 50 μL of protein extractionbuffer. The standard curve is produced for each new solution of 2,4-DNPH(the 2,4-DNPH solution is stable for 6 months in the dark). Theadvantage of this assay is its rapidity and simplicity, but it has thedefect of measuring the disappearance of substrate and not theappearance of product. In addition, the possibility of having falsepositives exists: a tyrosine amino transferase activity will give thesame result as the, HPPO activity. Specifically, in both cases, theketone function has disappeared. We therefore developed a rapid andsensitive HPLC method which makes it possible to confirm the productionof 4-HPA.

I..1.3.2—Activity Assay Analyzed by HPLC

An HPLC method was developed using a small Spherisorb ODS2 column,50×4.6 mm and particle size 3 μm. The chromatography is carried outunder isocratic conditions A: 90%; B: 10% (where buffer A: H₂O, 0.1% TFAand buffer B: acetonitrile), flow rate 0.8 mL.min⁻¹ and the elution isfollowed at 230 nm. Under these conditions, it is possible to separatethe 4-HPA, HGA, 3,4-DHPA and HPP in 5 minutes after injection. Thecolumn was custom made by Merck.

I.1.3.3—Purification of the Protein

The interests of simplicity were sought during the setting up of thisprotocol.

I.1.3.3.1—Preliminary Assays

The aim of the preliminary assays is to determine the influence ofcompounds (NaCl, KCl, 1-propanol, ethylene glycol, etc.) and of the pHon the enzyme activity. The reactions are carried out with crudeextracts of A. globiformis cultured on M^(Ag) medium containing tyrosineas the only carbon source (M^(Ag)-tyrosine). The test compound is addedto the reaction medium. To measure the influence of pH on the enzymeactivity of HPPO, various phosphate buffers are prepared.

I.1.3.3.2—Purification Protocol

The Arthrobacter globiformis strain is plated out on LB agar medium oron Columbia-ANC agar medium. After culturing for 16 hours at 29° C., acolony is removed and seeded in 5 mL of LB medium, under growthconditions for 8 hours at 29° C., 180 rpm. 50 μL of this preculture arethen inoculated into 1.5 L of M^(Ag)-tyrosine or M^(Ag)-HPP medium, andthe culturing is then carried out at 29° C., 180 rpm in Erlenmeyerflasks with thin rods (Belco). After culturing for 48 hours, the cellsare collected by centrifugation at 5000 g for 15 minutes at 4° C. Thecells are then resuspended in 50 mM Tris-HCl buffer, pH 7.4, and thencentrifuged as previously. The pellet is taken up in 2 mL of 50 mMTris-HCl buffer, pH 7.4. The cells are sonicated (Vibra Cell, SonicMaterials INC., Connecticut, USA) for 15 minutes, power 4, 30% pulse, inmelting ice. The insoluble debris are eliminated by centrifugation for25 min at 20 000 g, 4° C. The supernatant is recovered; it constitutesthe “crude extract”. It can be frozen in liquid nitrogen and then storedat −80° C. (for 6 months without apparent loss of activity). The crudeextract is loaded, without prior desalting, onto an “EMD/DEAE 650 S”weak anion exchange column (Merck) equilibrated in 50 mM phosphatebuffer, pH 7.4. Elution of the enzyme activity is obtained by applyingan NaCl concentration gradient (in solution in a 50 mM phosphate buffer,pH 7.4). The fractions containing the enzyme activity are pooled. Theprotein solution obtained is diluted 2.7-fold with 50 mM phosphatebuffer, pH 7.4. The proteins are then loaded onto a “source Q” stronganion exchange column (XK16, Pharmacia) (30 mL, Pharmacia)pre-equilibrated with a 50 mM phosphate buffer, pH 7.4. The proteinfractions of value, identified by the enzyme activity, are pooled andthen concentrated through UVIKON 10 kDa membrane. The resulting proteinextract is then desalted by the gel filtration technique using a “PD10”column (Pharmacia) equilibrated in 10 mM phosphate buffer pH 7.4, andeluted with this same buffer. The proteins are then loaded onto ahydroxyapatite column (XK9/15, Pharmacia) (2 mL; hydroxyapatite DNAgrade Bio-Gel® HTP gel; Bio-Rad) equilibrated with 10 mM phosphatebuffer, pH 7.4. The enzyme activity is eluted by applying a phosphategradient. The fractions containing the enzyme activity are pooled andconcentrated. The active proteins are conserved when the proteinconcentration is greater than 1 mg/mL by adding FAD, GSH and glycerol inorder to obtain the following final concentrations: 27 μM FAD, 110 μMGSH, 0.8% glycerol. The proteins thus prepared can be frozen at −80° C.for at least 6 months.

I.1.3.3.3—Assaying of Proteins

The proteins are assayed according to the Bradford method (1976) usingγ-globulin for the standard.

I.1.3.3.4—Staining of Protein Gels

The protein fractions are analyzed on 10% polyacrylamide gel accordingto the Laemmli method (1970). After migration, the proteins in the gelare stained either using the Coomassie Blue method (Chua, 1980) or usingthe silver nitrate method (Schoenle et al., 1984).

I.1.4—Protein Microsequencing of the N-terminal End and of InternalPeptides

The microsequencing of the protein is carried out using the Edman method(Laursen, 1971). To obtain the best results in the sequencing, the gelis prepared on the same day.

I.1.4.1—Preparation of the Acrylamide Gel and Electrophoresis thereof

The gels (8.5%, 10% or 12%) are prepared according to the Laemmli method(1970) using the Hoefer® minigel system. The proteins are diluted to onethird with a “denaturing loading blue” solution (150 mM Tris-HCl, pH6.8; 4% SDS; 2% (v/v) β-mercaptoethanol; 3.3% (v/v) glycerol; 0.03%bromophenol blue; qs 10 mL of milliQ water). After having been boiledfor 5 minutes, the proteins are loaded onto the acrylamide gel. Themigration is carried out at ambient temperature using a denaturingmigration buffer (25 mM Tris base; 250 mM glycine;0.014% (v/v)β-mercaptoethanol; 0.1% SDS) and applying a strength of 15 mA per gel.

I.1.4.2—Preparations for the sequencing of the N-terminal End

In order to be able to carry out the sequencing of the N-terminal end,the gel is transferred onto a PVDF membrane PROBLOTT®-AppliedBiosystems) using the semi-dry transfer technique. The electrotransferof the polypeptides is carried out in 30 minutes at 300 mA with the“Semy Thrv Plectroblotter” device (Bio-Rad) and in a CAPS-based medium(transfer buffer: 10 mM CAPS, pH 11.0; 10% (v/v) methanol). The transferbuffer contains no glycine which would risk “polluting” the sequencing.After the transfer, the membrane is rinsed for a few seconds with milliQwater. It is then immersed for a few seconds in a staining solutionbased on amido-black (Aldrich; ref.: 19.524-3). The solution consists of45% (v/v) methanol, 1% (v/v)-acetic acid, 0.1% (mlv) amido-black and63.9% (v/v) water. When the band corresponding to the protein ofinterest is visible, the membrane is thoroughly rinsed with milliQ waterand is then air-dried. The part of the membrane containing the proteinof interest (60 kDa) is cut out and sent for sequencing.

I.1.4.3—Preparations in View of Sequencing the Internal Peptides

In order to visualize the proteins in the gel, an amido-black stainingprotocol is used which is slightly different from that used to stain thePVDF membrane. After migration, the gel is fixed for two times thirtyminutes with a solution consisting of 50% methanol, 10% acetic acid, 40%milliQ water. The staining is carried out with a solution containing 45%methanol, 10% acetic acid, 45% water and 0.003% (w/v) amido-black. Theproteins appear gradually. When the staining is sufficient to locate theprotein, the gel is thoroughly rinsed with milliQ water. The band ofinterest is cut out and then dehydrated in a speed-vac (Savant). The gelband, having lost approximately a third of its length, is sent forsequencing. The internal peptides are obtained after digestion with theprotein with Lys-C endoprotease (sequencing grade, Boehringer). Theprotein in the polyacrylamide gel is digested in 150 μL of Tris-HClbuffer, pH 8.6 (0.1 M), containing 0.03% of SDS, at 35° C. for 18 hoursin the presence of 0.4 μg of Lys-C endoprotease. The digested protein isinjected onto a DEAE-C18 HPLC column (diameter 1 mm); the peptides areeluted using a gradient of acetonitrile (from 2 to 75%) containing 0.1%TFA. The Lys-C endoprotease specifically cleaves the polypeptides on thecarboxylic side of the lysines.

I.1.5.1—Theoretical Validation Using the Arthrobacter globiformis MnDGene

A portion (867 bp) of the MndD gene is amplified by using the primers“OZ- MndD-S711”: ACGTCACCGA AGAGGATGAA AAC (SEQ ID NO: 23) and“OZ-MndD-AS1578”: ACGGCCATTT CGGACTTTTC (SEQ ID NO: 24). The PCR iscarried out using the following program: 95° C. 5 min; 25 cycles: 95° C.45 sec, 56° C. 45 sec; 72° C. 1 min; 72° C. 5 min hold. The reactionmixture comprises 200 to 500 μL of dNTP, 20 to 200 ng of cosmid orgenomic DNA and 100 pmol of each primer in a final volume of 50 μL.

I.1.5.2—Identification by PCR of a Portion of the Gene Encoding the HPPOxidase

The PCR is carried out using the “Advantage®-GC-Genomic PCR” kit,(Clontech). This kit comprises, inter alia, a “GC melt” betaine-basedadjuvant and a mixture of thermoresistant polymerases—mainly withThermus thermophilus (Tth)-. The amplification is carried out on theArthrobacer globiformis genomic DNA, using the following program: 94° C.5 min; 30 cycles: 94° C. 20 sec, 60° C. 30 sec, 72° C. 3 min; 72° C. 6min; 4° C. on hold. The reaction conditions are 400 μM of dNTP, 50 ng ofgenomic DNA, 100 pmol of each primer and 1X “GC melt”, for a reactionvolume of 50 μL. Under these conditions, we amplify a band of 937 bpwhich we refer to as Z2.

The PCR amplification can also be carried out using Epicentre Tth or Tbr(Thermus brockianus—Finnzyme). Tbr is the only thermoresistantpolymerase tested to be able to carry out the PCR without additives(DMSO, glycerol, betaine); it is also a high-fidelity enzyme.

I.1.6—Screening of the Cosmid Library

The cosmid library is screened using the digoxigenin-labeled cold probetechnique (Boehringer Mannheim, 1995).

I.1.6.1—Preparation of the Z2-Dig Probe

The probe is labeled with digoxigenin by PCR in a final volume of 50 μL,under the conditions defined in paragraph II.5.2, except for the mixtureof dNTP consisting of: 90 μM dUTP-Dig; 135 μM dTTP; 225 μM dATP; 225 μMdCTP; 225 μM dGTP. The amplified probe is quantified by loading 3 μL ofthe reaction onto a 0.8% agarose gel. A slight background noise appears,i.e. the PCR is not sufficiently specific. In order to avoid allsubsequent problems, the entire PCR is loaded onto a gel and the band ofinterest is extracted using the Qiaex II kit (Qiagen).

I.1.6.2 —Transfer of the Cosmid Library onto Hybond N Membrane

The glycerol stock of the cosmid library prepared in E. coli HB101 isused to inoculate 2 mL of LBT¹⁵ medium. After growth for 8 hours, theOD₆₀₀ is estimated; sera dilutions are prepared in order to plate outapproximately 1000 clones per dish (144 cm²). After growth for 16 hoursat 37° C., the bacteria are transferred to Hybond N membranes (Amersham)and lysed according to Boehringer Mannheim's recommendations (1995). TheDNA released is fixed to the membrane by exposure to U.V. (120 mJdelivered in 45 sec—Stratalinker; Stratagene). The cell debris areremoved from the membranes by carrying out the proteinase K treatment asrecommended by Boehringer Mannheim (1995).

I.1.6.3—Prehybridization—Hybridization—Detection

The steps of prehybridization and hybridization are carried out in a bagplaced on a rocking platform, using the technique of hybridization withthe digoxigenin-labeled probe (Boehringer Mannheim, 1995). Theprehybridization 5×SSC; 0.5% SDS; 0.1% N-laurylsarcosine; 1% blockingagents (Boehringer Mannheim, ref.: 1096 176); 100 μg.mL⁻¹ sonicated anddenatured salmon sperm) is carried out for 0.4 hours at 65° C.Hybridization of the membrane is carried out overnight at 68° C. (freshprehybridization medium containing 20 ng.mL⁻¹ of digoxigenin-labeledprobe denatured for 5 min at 100° C.). The following day, the excessprobe and the aspecific hybridizations are removed with four washes withbuffer A (0.5×SSC; 0.1% SDS, 65° C.). The membranes are thenequilibrated for 5 min at ambient temperature in buffer B (138 mM malicacid, 142 mM NaCl, adjusted to pH 7.5 with sodium hydroxide pellets,0.3% tween 20). They are then saturated with blocking agents (BoebringerMannheim) for 30 minutes, before being hybridized with the alkalinephosphatase-coupled anti-digoxigenin antibody (“anti-digoxigenin-AP, Fabfragments”; Boehringer Mannheim) diluted to 1/10000 in a fresh solutionof blocking agents. After 30 minutes the membranes are rinsed for twotimes 15 minutes in buffer B, and then equilibrated for 5 minutes in thealkaline phosphatase reaction buffer (0.1 M Tris; 0.1 M NaCl; 0.05 MMgCl₂, pH 9.5). The membranes are covered with 1 ml of ready-to-use CSPDand are then incubated for 15 min at 37° C. This step at 37° C. allowsrapid activation of the alkaline phosphatase coupled to the antibody.The membranes are developed by exposing Hyperfilm® ECL (Amersham) for 1to 15 minutes.

I.1.6.4—Analysis of the Positive Cosmids by Southern and PCR

The cosmids identified in the hybridization on membrane are confirmed byPCR and by the Southern technique. In this case, the cosmid DNA,purified by alkaline lysis (Maniatis et al., 1982), is digested withrestriction enzymes and then separated on a 0.8% agarose gel. The gelsare transferred onto Hybond N⁺ membrane (Amersham) by the Southerntechnique in 20×SSC (Ausubel et al., 1995). After transfer, the membraneis rinsed with 2×SSC and the DNA is then fixed to the membrane usingU.V. (120 mJ delivered in 45 sec-Stratalinker; Stratagene). The membraneis then developed using the cold probe technique previously described.

I.1.7—Cloning Vectors and Host Bacteria

The PCR-amplified DNA sequences are generally cloned into the plasmidp-GEMT-easy (Proméga), which allows screening using the “blue-white”technique. For overexpression, the plasmid pKK223-3 (Pharmacia) is used,which places the gene under the control of a tac promoter. The cloningsare generally carried out using E. coli DH5α (New England Biolabs) or E.coli XL1 Blue (Stratagene). For overexpression, E. coli BL21 (DE3) willbe preferred.

I.1.8—Enzyme Activity of Acetolactate Synthase (ALS)

The acetolactate synthase (ALS) activity is measured using thecolorimetric method described by Chang and Duggleby (1997). Thereactions are carried out in microplates with a total volume of 250 μL.For each reaction, 25 μL of enzyme are incubated for 30 min at 37° C. in225 μL of reaction medium consisting of 50 mM KPi, pH 7.0; 50 mM sodiumpyruvate; 1 mM TPP; 10 mM MgCl₂; 10 μM FAD. The reaction is stopped byadding 25 μL of 10% H₂SO₄. The microplates are then incubated at 60° C.for 15 min 250 μL of 0.5% creatine and 250 μL of 5% α-naphthol in 4MNaOH (the α-naphthol solution should be prepared less than 10 min beforeuse) are then added. The microplate is then incubated for 15 minutes at60° C. and then 15 minutes at ambient temperature. A cherry red colorappears. The reading is carried out at 525 nm (ε_(m)=22 700 M⁻¹ cm⁻¹).

I.2—Results—Discussion

HPP oxidase is the first enzyme activity which we wish to introduce intothe plant in the context of creating the metabolic pathway bypassingHPPD. In order to be able to identify the gene encoding the HPP oxidaseactivity, various approaches were developed: (1) insertional mutagenesisand therefore identification of the gene through the loss of the enzymeactivity, (2) functional complementation of a microorganism using agenomic library, (3) purification of the protein in order to work backto the nucleic acid sequence. It is the third approach which waspreferred.

I.2.1—Purification of the HPPO

I.2.1.1—Optimization of the Culture Conditions

Before beginning to purify the protein, it is useful to determine whichculture conditions allow its expression in the bacterium. The results ofoptimization of the culture conditions show that the HPP oxidaseactivity is not detectable when the growth of A. globiformis depends ona carbon source such as succinate, fumarate or glucose. On the otherhand, the HPP oxidase activity is detected when A. globiformis iscultured using HPP, tyrosine or phenylalanine as the only carbon source.If the amount of yeast extract is increased (for example 200 mg.L⁻¹instead of 20 mg.L⁻¹), a decrease in the enzyme activity produced isobserved. The M^(Ag) medium is defined on the basis of theseobservations. Finally, it is observed that a high-density culture (atthe beginning of the stationary phase; OD₆₀₀˜1) exhibits a weaker HPPoxidase enzyme activity than in the case of a culture in the exponentialgrowth phase (OD₆₀₀˜0.4).

I.2.1.2—Preliminary Assays

We have just defined the optimal medium for the production of the HPPO,we now search for the conditions which do not impair the stability ofthe HPP oxidase during the purification processes. For thechromatographies involving anion exchange resins and thechromatographies as a function of pH, it is important to know thesensitivity of the enzyme to pH and to salts. We observe that theoptimum pH is between pH 7.0 and 7.8, as has been demonstrated byBlakley (1977). The enzyme appears to be relatively insensitive to salts(NaCl and KCl) since concentrations of greater than 750 mM are necessaryto observe a decrease in enzyme activity. We now know the conditions forgood expression of the enzyme activity and we have determined thesensitivity of the HPP oxidase activity to factors possibly interveningduring the purification. The purification of the HPPO can thereforebegin.

I.2.2.3—Purification of the HPPO

To purify the HPPO, the protocol described above is applied. The enzymeactivity is eluted from the DEAB EMD 650S with 150 to 200 mM of NaCl insolution in a 50 mM phosphate buffer, pH 7.4. The fractions containingthe enzyme activity are pooled and conserved overnight at 4° C. Freezingat this step in fact leads to a loss of activity. The proteins are thenloaded onto a Source Q resin. The enzyme activity is then eluted with anNaCl concentration of between 150 and 0.200 mM in solution in a 50 mMphosphate buffer, pH 7.4. The fractions containing the enzyme activityare pooled and then concentrated on UVIKON 10 kDa membrane, and storedat 4° C. overnight. Finally, the HPPO is purified in a third step byapplying a phosphate gradient to a hydroxyapatite column. The activityis eluted with a concentration of phosphate in the region of 30 mM. Thefractions containing the HPP oxidase enzyme activity, at thehydroxyapatite column outlet, are then analyzed on an SDS-8.5% PAGE gelstained with silver nitrate. The gel exhibits the development of twoprotein bands. By comparison between the enzyme activity profile and theprotein elution profile, we consider that the HPPO corresponds to thehigh molecular protein (approximately 60 kDa). In the attempt presented,the purification is initiated with 1.5 g of soluble proteins extractedfrom A. globiform is, and we recovered 150 μg of a mixture of proteins(including approximately 70 μg of HPPO). The purification factor interms of specific activity was not determined. As a result, we usedtotal reaction conditions to follow the elution of the enzyme activity.In addition, the problem was more the identification of the protein thanthe development of a purification protocol. The HPLC analysis, of thereactions carried out at the end of each purification step, shows theappearance of a product which has the same retention time as the 4-HPAstandard (SIGMA). Forty picomoles of the HPPO protein (60 kDa) aretransferred onto a PVDF membrane and are sent for sequencing at the sametime as 40 pmol of the protein included in the acrylamide gel. Theproteins transferred onto membranes serve to determine the N-terminalsequence, whereas the proteins included in the gel are used to determinethe sequence of internal peptides.

I.2.2.4—Results of Sequencing the HPPO

Few internal peptides are obtained on exiting HPLC, after digestion ofthe HPPO with Lys-C endoprotease. This result suggests that the proteincontains little lysine; specifically, Lys-C endopeptidase cleaves afterlysines. If lysine is relatively infrequent, digestion withendopeptidase K generates long peptide fragments which remain adsorbedin the column and cannot be eluted, even using very hydrophobicconditions. Based on the shape of the chromatographic peaks and also onthe apparent amount, three peptides were selected and then sequenced.They are named as a function of their order of leaving the HPLC column:peptide No. 4, peptide No. 6, peptide No. 11. Their sequence isrespectively: (A)WWAEALK (SEQ ID NO: 25), AAAGRILRLL DDAAGANASK (SEQ IDNO: 26), XDNRFTAVDF XT (where X is an undetermined amino acid) (SEQ IDNO: 27). The sequence of the first 30 N-terminal amino acids is obtainedwith an initial yield of 40%: TSLTVSGRVA QVLSSYVSD VFGVMGNGNV Y (SEQ IDNO: 28). The amino acid (methionine or valine) corresponding to theinitiation codon (ATG or GTG) is not found. The initial yield obtained(15 pmol BSA equivalent), compared with that obtained for the internalpeptides (30 to 35 pmol BSA equivalent), suggests that some of theproteins were blocked at the N-terminal. The N-terminal sequence and theinternal sequences obtained show no homology in the databases. Based onthe peptide sequences obtained, degenerate oligonucleotides aresynthesized in order to identify the HPPO gene by PCR.

I.2.3—Validation of the PCR Techniques and Identification of a Portionof the hppO Gene

I.2.3.1—Validation of the PCR Techniques

The content of guanine and cytosine base (SC %) of the majority ofArthrobacter sp. genomic DNAs is between 59 and 66%; however, it is 67to 69% for A. agilis (formerly Micrococcus agilis) (Koch et al., 1995),70% for A. atrocyaneus (Jones et al., 1991) and 73% for an Arthrobactersp. identified in arctic ices (Junge et al., 1998). These high contentsof guanine and cytosine can make it more difficult to carry out PCR. Forthis reason, we validated our PCR methods (genomic DNA, polymerases,etc) using the gene encoding Arthrobacter globiformis “Manganesedependent Dioxigenase” (MndD) (Boldt et al., 1995). This enzyme of theHPP degradation pathway catalyzes the opening of the aromatic ring of3,4-dihydroxyphenyl acetate. For the control amplification of the MndDgene, we tested thermophilus aquaticus (Taq) thermoresistant polymerasesmarketed by various suppliers (Perkin Elmer, ATGC, Appligène, Qiagen,Sigma). In all cases, amplification of the MndD gene is obtained.However, under equivalent conditions, using the degenerate primersencoding the HPPO peptides, amplification of the hppO gene is notobtained even using additives (DMSO, glycerol).

I.2.3.2—Identification by PCR of the N-terminal Portion of the hppO GeneWe specifically amplified a 936 bp DNA sequence which might correspondto the N-terminal portion of the hppO gene. The amplification wasobtained using, firstly, the degenerate primers O×3: TTNGCNCCNGCNGCRTCRTC (SEQ ID NO: 29) and OZ10N: GAYGTNTTYG GNGTNATGGG NAAYGG (SEQID NO: 30) corresponding, respectively, to a portion of peptide No. 6and to a portion of the N-terminal peptide sequence and, secondly, the“Advantage GC Genomic PCR” kit (Clontech). The Clontech kit is designedto carry out PCRs on GC-base-rich genomes. It contains a mixture ofthermoresistant polymerases (including Tth) and a betaine-basedadditive. Tth is a thermoresistant polymerase purified from Thermusthermophilus. The degeneracy of each primer is 1024, i.e. one primer outof 1024 exhibits the exact nucleic acid sequence of the gene beingsought. The degeneracy originates from the fact that an amino acid canbe encoded by several codons; for example, alanine is encoded by fourcodons (GCA, GCC, GCG, CGT). The degeneracy code used for the primers isdefined as follows: N=A or T or G or C; R=A or G; Y=T or C. Thetheoretical hybridization temperatures are, respectively, 55.4° C. and57.6° C. Despite a hybridization temperature of 60° C. used in the PCR,the OX3 primer alone allows nonspecific amplifications. We specificallyamplified, by PCR, a 936 bp DNA fragment, using two degenerate primers.We must be sure that this amplified DNA corresponds correctly to thehppO gene being sought.I.2.4—Characteristic of the 936 bp DNA Fragment

The 936 bp DNA fragment amplified by PCR is purified on agarose gel. Itis then cloned into pGEM-T easy, according to the supplier'sinstructions, and then sequenced. When the nucleic acid sequenceobtained is translated, it is observed that it encodes, at the two ends,for the entire peptide No. 6 and for a large part of the N-terminalsequence. We are therefore sure to have amplified a portion of the geneencoding the purified and microsequenced protein, the HPPO. The nucleicacid sequence contains 73% of guanine (G) and cytosine (C) bases; thepossible formation of secondary “stem-loop” structures is also noted inthe first 250 bases of the messenger RNA. This high content of G and Cbases and also the existence of the secondary structures may partlyexplain the difficulties encountered in achieving the PCR amplificationof part of this gene. The 936 bp nucleic acid sequence and also thecorresponding protein sequence exhibit no homologies with the sequencesrecorded in the databases. We now have a 936 bp sequence oriented fromthe N-terminal toward internal peptide No. 6. Since the protein isapproximately 60 kDa, a gene of approximately 1650 bp is sought. Thereremains therefore approximately 700 bp to be identified. For this, wewill screen the A. globiformis genomic library produced in the cosmidpLAFR5 and amplified in E. coli HB101.

I.2.5—Screening of the A. globifomis Cosmid Library

The genomic library prepared is transferred onto membranes and is thenscreened using, as probe, the 936 bp DNA fragment labeled withdigoxigenin. The standard protocol is adapted for a “conventional” DNA(60% AT), while the 936 bp fragment exhibits an estimated proportion of23% AT. If we keep the same dUTP-Dig/dTTP ratio as in the case of aconventional DNA, we obtain a weakly labeled probe and therefore a lesssensitive detection. We therefore optimize the dUTP-Dig/dTTP proportionnecessary for labeling the probe (paragraph II.7.1). Screening of thegenomic library made it possible to identify four cosmids (Cos1A, Cos2A,Cos4A, Cos17A1) having different restriction profiles. By comparing theresults of Southern hybridization obtained using the cosmids with thoseobtained using the Arthrobacter globiformis genomic DNA, we selected thecosmid 2A. FIG. No. 14 illustrates the approach used taking as anexample digestion of the cosmids with the Not I restriction enzyme. Itis observed first of all that the cosmid vector pLAFR5, digested withNot I, does not hybridize with the Z2-Dig probe. On the other hand, itis observed that the cosmid 1A exhibits a single hybridization band at2.3 kb while, the cosmids 2A, 4A and 17A exhibit two hybridization bandsat 4.3 and 2.3 kb. Now, digestion of the A. globiformis genome with NotI produces two bands of 4.3 and 2.3 kb; as a result, we consider thatthe cosmid 1A does not contain all the information sought. Based onother restrictions and using an equivalent approach, the cosmids 4A and17A, are eliminated. The cosmid 2A is then sequenced over a distance ofapproximately 3 kb on either side of the Not I site identified in themiddle of the Z2-Dig probe. The results of hybridization of the genomicDNA also show that the gene is present in a single copy. We haveidentified the cosmid 2A which we have sequenced over 6.2 kb. We willnow be able to analyze this DNA sequence derived from the Arthrobacterglobiformis genome.

I.2.6—Overall Analysis of the 6.2 kb of Arthrobacter globifoxmis GenomicDNA

Using Vector Nti software, the position of the potential genes isdefined from the nucleic acid sequence of 6255 bp obtained by sequencingthe cosmid 2A. The 936 bp sequence, identified by PCR, is found to bepart of a potential gene. This potential gene therefore probablycorresponds to the hppO gene. Four other genes (A, B, C, D) arepotentially identified (FIG. 3) by carrying out a search by homologyusing the BLASTX algorithm. Gene A will encode an amino acidtransporter, Gene B will encode a histidinol-phosphate aminotransferase; however, previous studies show that this enzyme hastyrosine amino transferase activity in the Gram-positive bacteriumBacillus subtilis (Nester & Montoya, 1976) gene C will encode atranscription regulator, while gene D will encode an operon regulator.

I.2.7—Analysis of the hppO Gene

I.2.7.1—General Description

Over the 6256 bp sequence obtained, the hppO gene (in green) isdelimited in 5′ by the ATG initiation codon at position 3143 and in 3′by the stop codon TAG (in red) at position 4823. The gene therefore hasa real length of 1680 bp. It exhibits a high content of G and C bases(71.4%, GC). The search for homologies in the nucleic acid sequences(BLASTN) gives no identification. In order to more thoroughlycharacterize the gene, the specific elements of transcription and oftranslation are sought.

I.2.7.2—Elements Characterizing the Transcription and Translation of thehppO Gene

The potential transcription promoter boxes are identified (FIG. 4). Box“−10”, termed “Pribnow box” is located between 3082 to 3088 (AAAAATA)and box “−35” is located at position 3055 to 3059 (TTGCA). These boxesthus defined are slightly different from the canonic sequences(respectively TATAAT and TTGACA; Singer & Berg, 1992). This may reflecta weak interaction with the factors allowing constitutive transcriptionor else the necessary interaction with different transcription factors.The adenine at position 3096 might be the transcription initiation base.Finally, a sequence corresponding to the binding site for the CAPprotein (catabolic gene activator protein) is identified betweenpositions 3068 to 3072 (TGTGA). Finding this CAP protein-binding site isin agreement with the results obtained in the optimization of theculture conditions. In conclusion, the transcription of the hppO gene isprobably under the control of a weak promoter, in particular regulatedby glucose. The Shine-Dalgarno sequence (Singer & Berg, 1992) allowsbinding of the ribosomal small subunit. It is identified (GACGAT; atpositions 3131 to 3136) 12 bases upstream of the translation initiationcodon (ATG), by analogy with the AGGA consensus sequence. It is alsoobserved that the 5′ terminal portion (approximately 250 bases) of themessenger RNA is capable of forming a stem-loop structure. Now, thesecondary structure of the region of the mRNA which is in the region ofthe initiator ATG influences the translation initiation step. Thus, theinitiation is zero or relatively inefficient when the initiator ATG orthe Shine-Dalgarno sequence is involved in intramolecular pairing. Thequestion may therefore be posed of whether the step-loop structuresobserved have a possible role in regulating the translation.

I.1.2.7.3—Expression of ht e HPPO Under the Control of the Tac Promoter

Overexpression of the HPPO is advantageous for defining the kineticcharacteristics, to allow the production of antibodies, but also for thepurpose of structural analysis. The gene is cloned into a vectorpKK223-3 in two stages. The gene, amplified by PCR under the conditionsdefined for the identification of the hppO gene and using the primersHPP-N-sense (CATGACTTCA CTTACAGTGT CC) (SEO ID NO: 31) and HPP-C-term(CAAACTGAGT AGCAGCTCAG G) (SEO ID NO: 32), is cloned into the vectorpGEMT-easy. The clone exhibiting the hppO gene in the antisensedirection with respect to the lac promoter is selected. It is thendigested with Eco RI. By doing this, the hppO gene is recovered, and isinserted into the vector pKK223-3 digested with Eco RI. The clonepKK3-2, exhibiting the hppO gene under the control of the tac promoteris selected. When the expression of the clone pKK3-2 is induced byadding IPTG, HPP oxidase activity can be detected. However, theoverexpressed protein (57.4 kDa) cannot be detected in a crude extractseparated on denaturing acrylamide gel. The overexpression protocoltherefore remains to be improved. We also envision cloning the HPPO as afusion with a Tag sequence (GST, polyhistidine, protein A, etc) in orderto facilitate purification of the overexpressed protein. We have justdefinitively shown that the identified gene encodes an HPP oxidaseactivity. However, in carrylng out homology searches at the proteinsequence level (BLASTX or BLASTP), it is observed that the HPPO proteinexhibits up to 25% identity with acetolactate synthases (ALSs), pyruvateoxidases (POXs) and pyruvate dehydrogenases (PDHs). It is thus possibleto identify very conserved motifs such as those regarding TPP cofactorbinding (FIG. 5). In addition, the hydrophobicity profile of the HPPO isvery close to that obtained for ALSs (not shown). In order to be surethat the identified gene really encodes the HPPO and not an ALS, a POXor a PDH having a secondary activity of the HPP oxidase type, we decidedto test the HPPO for a possible secondary activity.

I.2.8 HPPO Versus ALS

The protein homology searches show that HPPO exhibits up to 25% identitywith ALSs. This result, although initially surprising, has a certainlogic. Specifically, these two enzymes use FAD and TPP as reactioncofactors. They both carry out a decarboxylation. Moreover, one of thesubstrates of ALS is pyruvate; now, our substrate is a β-substitutedpyruvate: hydroxyphenyl pyruvate. It is therefore possible that thestructure of the active site is close and that, consequently, theseproteins share common enzyme activities. We used the recombinant largesubunit and purified ALSs from Arabidopsis thaliana (Chang & Duggleby,1997) and from E. coli (Hill & Duggleby, 1998) to serve as a positivecontrol in our experiments carried out in order to search for ALSactivity in the HPPO. The results obtained show that the HPPO does notexhibit any ALS activity. We show on this occasion that the two ALSstested have no HPP oxidase activity. Finally, we observe that the HPPOis not inhibited by 115 ppm of imazapyr (ALS inhibitor, cyanamid). Theseresults clearly show that, despite common points (protein sequence andhydrophobicity), ALSs and the HPPO are clearly different enzymes whichdo not have secondary enzyme activities.

EXAMPLE 2 Identification of the Genes Encoding 4-HPA 1-Hydroxylase

4-HPA 1-hydroxylase (HPAH) converts 4-HPA to HGA via a hydroxylationreaction accompanied by displacement of the acetyl chain. Its activityhas been characterized on crude extracts of Rhodococcus erythropolis S1(Suembri et al., 1995) or on partially purified extracts of P.acidovorans (Hareland, 1975). It was purified by Suemori et al. (1996),but the protein and gene sequences are not published. In order to beable to introduce this enzyme activity into the plant, it is necessaryto identify the gene.

Various approaches can be envisioned: (1) phenotypic and/or functionalcomplementation using a genomic library, (2) insertional mutation andtherefore identification of the gene through the loss of the enzymeactivity, (3) purification of the protein in order to work back to thenucleic acid sequence. We chose to develop these three approaches withPseudomonas acidovorans because there are many molecular biology toolswhose effectiveness has been demonstrated on various species and strainsof Pseudomonas. By way of examples, mention may be made of the mini-Tn5transposon (De Lorenzo et al., 1990), the broad host spectrum vectorssuch as pBBR1MCS (Kovach et al., 1994, 1995; D'Souza et al., 2000), andthe techniques for transfer by conjugation., The mini-Tn5 transposon canbe used either to disturb a gene (de Lorenzo et al., 1990; Fedi et al.,1996; Campos-Garcia et al., 2000) or to introduce a gene into thebacterial genome (Prieto et al., 1999). We began with the approach byphenotypic, complementation because this appeared to be the most rapidand the most simple. This approach was followed by the two othersimultaneously.

However, we will not tackle the approach by insertional mutagenesis heresince this approach was not subsequently exploited.

II.1—Materials and Methods

II.1.1—Construction of a P. acidovorans. Genomic Library in E. coli

To construct the library we used the cosmid pLAFR5 and the genomic DNAof P. acidovorans. We used the host strain E. coli HB101.

II.1.2.1—Purification of the 4-HPA 1-Hydroxylase

In the reaction catalyzed by 4-HPA 1-hydroxylase, described by Harelandet al. (1975), molecular oxygen and NADH,H⁺ are consumed. We chose tomeasure the enzyme activity by following the oxidation of NADH,H⁺ toNAD⁺. The reaction medium comprises: 300 μM NADH,H⁺; 6.7 μM FAD; 100 mMKPi; 1 mM DDT; 10 to 50 μg of proteins. The reaction is triggered byadding the substrate: 1 mM 4-HPA. The reaction is followed at 340 nm orat 292 nm, for 2 to 10 min. Specifically, the consumption of NADH,H⁺results in a decrease in absorbance at 340 nm, while the production ofhomogentisate results in an increase in absorbance at 292 nm. Thespectrophotometric assay is very rapid, it is used routinely to followprotein elution in purification steps.

II.1.2.1—HPLC Activity Assay

Analysis of the enzyme reactions by HPLC makes it possible to confirmthe production of HGA (retention time, UV spectrum). The enzyme assay iscarried out under the same conditions as above. However, the reaction isstopped by adding a third of a volume of 20% perchloric acid. Thereactions are then analyzed by HPLC using isocratic elution with 90% ofphase A and 10% of phase B or 92% of phase A and 8% of phase B. Phase Ais milliQ water containing 0.1% of trifluoroacetic acid (TFA) and phaseB corresponds to acetonitrile. In the 90%-10% isocratic elution, the HGAis eluted in 1.2 min whereas in the 92%-8% isocratic system, it iseluted in 1.4 min. The elution is generally recorded at 230 nm. Van denTweel et al. (1986) used 2,2′-bipyridyl (non-heme iron proteininhibitor) to inhibit the homogentisate dioxygenase and thus allowaccumulation of the HGA. For this reason, 2 mM of 2,2-bipyridyl is addedto certain reaction media. Under these chromatographic conditions, it ispossible to identify the 4-HPA and the HGA. The HPLC system consists ofan Alliance 2690 HPLC (Waters) and a 996 diode array detector (Waters).

II.1.2.3—Purification of the HPAH Protein

Pseudomonas acidovorans is cultured for 48 hours on M63 mediumcontaining 4-HPA as the only carbon source, at 29° C. 220 rpm. Thebacteria are centrifuged at 3000 g for 15 min at 6° C. (Beckmann J2/21M/E centrifuge). The bacterial pellet is taken up in the sonicationbuffer (0.1 M KPi, pH 7.2; 1 mM MgSO₄; 1 mM DTT; 1 mM benzamidinehydrochloride; 5 mM caproic acid). Benzamidine hydrochloride and caproicacid are protease inhibitors. The sonication is carried out for 9minutes, sonicating every forty seconds for twenty seconds at power 5(Vibra Cell, Sonic Materials INC., Connecticut, USA). During thesonication, the sample is kept at the temperature of melting ice. Thesonicated extract is centrifuged at 15 000 g for 15 min at 4° C. Thesupernatant recovered is precipitated with 1% of streptomycin sulfate.The precipitate is eliminated by centrifugation at 15 000 g for 15 minat 4° C. The supematant is desalified on a PD10 column (Pharmacia) andthen loaded onto a DEAE/EMD 650 S column equilibrated in buffer A (20 mMKPi, pH 7.2, 10% glycerol, 1 mM MgSO₄, 1 mM DTT). The elution is carriedout using a buffer B (buffer A; 1 M KCl; 100 μM FAD). The 4-HPA1-hydroxylase activity is eluted for a KCl concentration in the regionof 150 mM. The active fractions, concentrated through UVIKON 10 kDamembrane and then desalified on a PD 10 column, are then loaded onto aRed affinity column (Red 120 Agarose type 3000 CL, SIGMA Ref R-0503)equilibrated in buffer A (above). The elution is carried out in twostages. The first is washing of the Red colum using buffer A enrichedwith FAD at a final concentration of 50 μM. The second alllows elutionof the protein; for this, buffer A enriched is FAD (3 mM) and in NADH,H⁺(10 mM). The fractions, containing the protein of interest, are pooled,concentrated and frozen at −80° C.

II.1.3—Protein Microsequencing of the N-terminal End and of InternalPeptides

The same protocol as that described in the case of the HPP oxidase wasused to carry out the sequencing of the purified protein. However, inorder to produce the internal peptides, the protein was digested withtrypsin instead of Lys-C endopeptidase. Trypsin cleaves after argininesand lysins. Digestion with trypsin generally leads to the production offragments which are smaller than those obtained in a digestion withLys-C endopeptidase. In order to be able to sequence with precision therecovered peptides, it is sometimes necessary to repurify the recoveredpeptides by HPLC.

II.1.4—Identification of a Portion of the Gene Encoding the HPAH byDegenerate PCR

The degeneracy code given on page 43 is used for the synthesis of thedegenerate primers. The PCR is carried out in a final volume 50 μL, in200 μL tubes. The reaction solution contains the Perkin Elmer buffer,250 μM dNTP, 50 ng of P. acidovorans genomic DNA, and 2 enzyme units ofAmpliTaq (Perkin Elmer). The reaction is carried out using a “HybaidTouchdown” thermocycler: 3 min at 94° C., then forty five cycles: 30 secat 94° C., 1 min at 50° C., 1 min 30 sec at 72° C., followed by a finalelongation of 5 min at 72° C. before returning to 4° C., The PCR isevaluated after loading 10 μL onto a 1% agarose gel. Under theseconditions, a 536 bp band is identified.

II.1.5—Screening of the P. acidovorans Cosmid Library

The cosmid library is plated out on LBT¹⁵ medium and allowed to grow for16 h at 37° C. The dishes are then transferred to 4° C. After one hour,the colonies are transferred to Hybond N membranes (Amersham) accordingto the method of Grunstein & Hogness (1975). The membranes arehybridized using the 536 bp PCR fragment previously identified andpurified. Detection is carried out with ³²P. The probe is labeled usingthe “DNA Ready to Go” kit (Pharmacia). The prehybridization,hybridization and washing are carried out in vials. The membranes areprehybridized in a solution composed of 5×SSC, 6% Denhardt's and 0.5%SDS, for 4 hours at 68° C. The hybridization is carried out for 16 hoursat 68° C. The washes are carried out at 65° C. in 2×SSC, 0.1% SDS. Themembranes are developed by exposing Kodak or Amersham films.

II.1.6—P. putida Growth Media

Pseudomonas putida is cultured on Luria-Bertani (LB) or 2YT rich mediumcontaining 100 μg.mL⁻¹ of rifampicin. Other antibiotics are added asneeded (example: tetracyclin 15 μg.mL⁻¹). The minimum medium M63containing 1.5 g.L⁻¹ of 4-HPA as the only carbon source is used to testthe functional complementation. In this case, the antibiotics areomitted. All the cultures are prepared at 29° C.

II.1.7—Transformation of P. putida by electroporation

1 liter of LB Rifampicin (100 μg.mL⁻¹) medium is inoculated with aculture of P. putida grown at 29° C. for approximately 16 hours withshaking at. 180 rpm. When the OD_(600nm) is in the region of 1.2, thecells are collected by centrifugation for 15 min at 3000 g, 4° C. Theculture medium is removed and the cells are taken up with 400 mL of 10%glycerol at 4° C. The cells are centrifuged once again at 3000 g for 20min at 4° C. Two further washing steps are carried out with,respectively, 200 then 100 mL of 10% glycerol at 4° C. Finally, thebacteria are taken up with 3 to 10 mL of 10% glycerol and thendistributed into 100 μL aliquots which are immediately frozen in liquidnitrogen. The bacteria thus prepared are conserved for at least sixmonths at −80° C. During the preparation, a loss of bacteria due tolysis is observed. The cosmid (Tet^(R)) DNA is introduced into the P.putida (Rif^(R)) by electroporation. The electroporation (Bio-Rad GenePulser^(TM)) of 80 ng of cosmid DNA into 100 μL of electrocompetent P.putida is carried out in a 2 min electroporation cuvette under a voltageof 0.9 volts with an electroporator resistance of 200 Ω. Under theseconditions, the time constant τ is approximately 4.5 msec. After theelectric shock, the cells are taken up with 900 μL of LB and culturedfor 1 h 30 at 29° C., 180 rpm. The transformed P. putida are selected onLB Ri¹⁰⁰ Tet¹⁵ agar medium.

II.1.8—Modification of the Broad Host Spectrum Vector pBBR1MCS-Gm^(R)

We used the broad Gram-negative host spectrum vectors of the pBBRlMCSseries (Kovach et al., 1994, 1995). These plasmids, which have aBordetella bronchiseptica origin of replication replicate atapproximately 20-30 copies per cell in E. coli. They contain two Not Isites. In order to facilitate the subsequent clonings, the Not I sitepresent outside the multiple cloning site (MCS) on the plasmidpBBR1MCS-Gn^(R) is deleted. For this, the plasmid is cleaved with Sfi I(50° C.) and then treated with T4 DNA polymerase in order to obtainblunt ends. The plasmid is religated on itself (T4 DNA Ligase -NewEngland Biolabs). After ligation (16 hours, 16° C.), a digestion withSfi I is carried out in order to eliminate the possible “wild-type”plasmids, and then E. coli DH5α are electroporated. The plasmid DNA isisolated from the clones selected on LB Gm²⁰ medium. The plasmid DNAsare characterized with two digestions: Not I and Not I/Bgl II. A cloneis selected: pBBR1MCS-Gm-Not-U.

II.1.9—Subcloning of Ccos8 in PBBR1MCS-Gm-U

The cosmid Ccos8 is restricted with Not I and then loaded onto anagarose gel. After migration, 6 DNA bands are visualized: 1.7; 3; 4; 5;8; 10 kbp. The bands are purified with Quiaex II. Moreover,pBBR1MCS-Gm-Not-U is restricted with Not I, and dephosphorylated usingshrimp alkaline phosphatase (SAP). The various bands are then ligated(T4 DNA ligase, 16 hours, 16° C.) into the vector using varying“insert/vector” ratios. E. coli DH5α are transformed with the ligationproducts.

II.1.10—Triparental Conjugation Between E. coil and P. putida

In order to transfer the various Ccos8 (Gm^(R)) subclones from E. coliDH5α to P. putida (Rif^(R)), triparenteral conjugation is carried out ona filter using the protocol described by De Lorenzo et al. (1990). Thebacteria recovered are plated out on LB Rif¹⁰⁰ Gm²⁰ and on M63 having4-HPA as the only carbon source.

II.1.11—Elimination of the Plasmid p5kbC

In order to rapidly eliminate the plasmid p5kbC of P. putida, theincompatible origins of replication strategy is used, and the loss ofp5kbC is forced using antibiotics. P. putida (Rif¹⁰⁰) complemented withthe plasmid p5kbC (Gm^(R)) is transformed with pBBR1MCS Kn^(R). Theclones obtained (Rif¹⁰⁰ Gm^(R) Kn^(R)) are verified for theircomplementation activity. The clones are then cultured on two media: LBRif¹⁰⁰ Kn¹⁵⁰ Gm²⁰ and LB Rif¹⁰⁰ Kn¹⁵⁰. In doing this, the selectionpressure for p5kbC and pBBR1MCS KnR or else only for pBBR1MCS Kn^(R) ismaintained. Growth is carried out at 29° C. The subculturing is carriedout every three days. At the eighth subculturing, the colonies aresubcultured on 4 different media (M63, M63+4-HPA, LB Rif¹⁰⁰ Kn¹⁵⁰ Gm²⁰and LB Rif¹⁰⁰ Kn¹⁵⁰) whatever the dish of origin. The state growth isthen recorded after 2 and 7 days.

II.1.12—Identification, of the Proteins Contributing to the EnzymeActivity

II.1.12.1—Preparation of Crude Extracts of P. putida

Two P. putida clones are cultured on LB Gm²⁰ for 24 hours. The firstcomprises the plasmid pBBR1MCS-Gm-Not-U, while the second contains thecomplementation plasmid p5kbC. After sonication in a buffer (0.1 M KPi;1 mM MgSO₄; 1 mM DTT; 1 mM benzamidine hydrochloride; 5 mM caproicacid), then centrifugation at 20 000 g for 10 min at 4° C., thesupernatant is tested for its 4-HPA 1-hydroxylase activity using the twomethods for measuring enzyme activity. The crude extracts are alsoanalyzed by SDS-10% PAGE.

II.1.12.2—Transfer onto Membrane, N-terminal Sequencing

The sequencing is carried out as in Example I.

II.1.12.3—S75 Gel Filtration

The eluate (5 mL) is concentrated 10-fold using a 10 K Macrosep™ (PallFiltron) for two hours at 4° C. The concentrated 500 μL are injectedonto a Superdex™75 prep grade gel filtration column (HiLoad 16/60,Pharmacia) pre-equilibrated with 700 mL of buffer (0.02 M KPi, pH 7.2;10% glycerol; 1mM MgSO₄; 1 mM DTT; 4° C.) at a flow rate of 0.7mL.min⁻¹. The chromatography is carried out at 4° C. with a flow rate of1 mL.min⁻¹. The fractions are collected every minute and stored at 4° C.

II.1.12.4—Construction of pBBR1MCS FT12Δ1

To construct the plasmid pBBR1MCS FT12Δ1, a two-step cloning strategy isused. The plasmid p5kbC is digested with Nsi I and Not I. The insertobtained, encoding genes 1, hpaH and 3, is then cloned intopBBR1MCS-Gm-Not-U digested with Ps I and Not I. The resulting clone,named pBBR1MCS FT12, is restricted with Hind III and Asc I, thenblunt-ended and, finally, religated. In doing this, genes 1 and 3 aredestroyed and the hpaH gene is under the control of the lac promoter ofthe original vector. The plasmid pBBR1MCS FT12Δ1 is thus obtained (FIG.6).

II.1.12.1—Construction of pL1lac2

The laboratory possesses a plasmid named “Clone L”. This constructcorresponds to the cloning of the P. fluorescens HPPD gene promoter intothe vector pBBR1MCS-Kn^(R). The HPPD gene promoter is functional in P.putida and in E. coli. The plasmid “Clone L” is digested with Bam HI andHin dIII, which makes it possible to recover the insert containing thepromoter and the HPPD gene of P. fluorescens. This insert is thenligated into the vector pBBR1MCS-Gm^(R) digested with Bam HI and HindIII. The resulting clone is named pBBRG-L-HPPD. The plasmid obtained,digested with Nco I to remove the gene encoding HPPD, is ligated withthe hpaC gene amplified by PCR and digested with Aft III. The constructobtained is called pBBRG-L-ORF1. To amplify the hpaC gene by PCR,primers which make it possible to introduce an Aft III site at thebeginning and at the end of the gene (the Aft III site is compatiblewith the Not I site) are used. The primers used are: positioned 5′ ofthe gene: GCAGGATGCA CATGTCCACC AAGAC (SEQ ID NO: 33) and positioned 3′of the gene: CGGACGCCGA CATGTATCAG CCTTC (SEQ ID NO: 34). The PCR iscarried out using 1 unit of KlenTaq polymerase (Sigma), 250 μM of dNTP,200 nM of each primer and 50 ng of the plasmid p5 kbC. The PCR programis defined as follows, on a Perkin Elmer 9600: 3 min at 95° C.; then 20cycles: 94° C. for 1 min 60° C. for 30 sec, 68° C. for 3 min; finally, alast step of 10 min at 68° C. is carried out. The plasmid pBBR1MCSFT12Δ1 previously obtained is restricted with Ssp I and Not I. The Not Isite is blunt-ended by treatment with Pfu. The fragment recovered (2468bp), containing the hpaH gene under the control of the lac promoter, isligated into pBBRG-L-ORF1 digested with Ssp I. The clone containing thehpaC gene and hpaH gene in the antisense direction is selected and isnamed pL1lac2. All this cloning is carried out in E. coli DH5α.

II.2—Results

Various approaches can be envisioned for identifying the gene encodingthe 4-HPA 1-hydroxylase activity of P. acidovorans. We decided initiallyto use an approach by phenotypic coloration. This approach appears to besimple and rapid. We in fact possess in the laboratory a phenotypicscreening tool for detecting the production of HGA. Now, the enzymebeing sought converts 4-HPA to HGA.

II.2.1—Approach by Phenotypic Coloration

We have observed in the laboratory that E. coli K12 cannot grow usingtyrosine or 4-HPA as the only carbon source. In addition, we know thatE. coli K12 has tyrosine aminotransferase activity which allowssynthesis of tyrosine from HPP. This enzyme activity is reversible, andthe cell can therefore produce HPP from tyrosine. If the rich culturemedium is enriched in tyrosine (1 g.L⁻¹), the tyrosine is imported intothe bacteria, which accumulate it and then convert it to HPP, accordingto the equilibrium constant for the conversion reaction between HPP andtyrosine. In the laboratory, we have already observed that, if weintroduce the P. fluorescens HPPD into E. coli K12, then the HPPproduced during the deamination of tyrosine is converted intohomogentisate (HGA). Since the reaction catalyzed by the HPPD isirreversible, the HGA accumulates in the cell, where it is oxidized thenpolymerizes spontaneously to form an ochronotic pigment which is brownin color. This therefore gives us a means of detecting the production ofHGA. The 4-HPA 1-hydroxylase being sought converts 4-HPA to HGA. The E.coli HB101 containing the Pseudomonas acidovorans genomic library aretherefore plated out on 2YT agar medium enriched in 4-HPA. After twodays, two colonies become brown: they therefore produce homogentisate.However, the enzyme activities detected on the crude extracts of thesetwo clones reveal enzyme activity of the HPPD type whereas the 4-HPA1-hydroxylase activity sought is discrete, or even nonexistent. Apriori, this approach made it possible to select the clones for whichthe cosmid contains the gene encoding a P. acidovorans HPPD and not the4-HPA 1-hydroxylase. In the in vitro preliminary study on the crudeextracts of P. acidovorans, the HPPD activity was not identified. It maybe presumed that the P. acidovorans HPPD activity would be expressedwhen the bacterium is cultured on rich medium, whereas the 4-HPA1-hydroxylase activity would be expressed when 4-HPA is the only carbonsource. Since this approach did not make it possible to identify the4-HPA 1-hydroxylase, we decided to purify the enzyme. Once the proteinis identified, it is possible to work back to the corresponding gene.

II.2.2—Purification of the 4-HPA 1-Hydroxylase

In order to follow the purification of the protein, its 4-HPA-dependentNADH,H⁺ oxidase activity is assayed. The protein is thus purified tovirtual homogeneity by applying the purification protocol describedabove. The enrichment factor for the specific NADH,H⁺ oxidase activityis generally between 50 and 100 depending on the preparations. OnSDS-PAGE, the protein has an apparent molecular weight of 60 kDa. Infact, it is observed that the NADH,H⁺ oxidase activity and theproduction of HGA are visible on leaving DEAE/EMD 650S. On the otherhand, on leaving an affinity column, the production of HGA is verydifficult to detect; the NADH,H⁺ oxidase activity remains, however,dependent on 4-HPA being added to the reaction medium. If the hypothesisthat the enzyme is monomeric is taken as a basis, the loss of catalyticactivity allowing the production of HGA can be explained by supposingthat a part of the protein has been damaged (for example: loss of astrongly associated cofactor) during its passage through the Red column.The site catalyzing the oxidation of NADH,H⁺ would not be affected. Itmay also be supposed that the enzyme sought is a heterodimer. The lossof catalytic activity would then be explained by the loss of the monomerresponsible for the production of HGA. Many heterodimeric flavinmonooxygenases have been identified in the literature, all having anaromatic substrate, in varied bacterial species (Adachi et al., 1964;Arunachalam et al., 1992, 1994; Prieto et al., 1993; Prieto & Garcia,1994; Arunachalam & Massey, 1994; Takizawa et al., 1995; Xun, 1996; Xun& Sandvik, 2000). However, two hypotheses exist to explain the functionof these heterodimeric enzymes:

(1) Arunachalam et al. (1992, 1994) proposed that the4-hydroxyphenylacetate 3-hydroxylase of P. putida consists of a 65 kDahomodimeric flavoprotein and also a 38.5 kDa coupling protein. Theflavoprotein alone is capable of oxidizing NADH,H⁺ independently of thepresence of 4-HPA. This oxidation of NADH,H⁺ makes it possible to renewthe NAD⁺ “pool”, but produces H₂O₂ in stoichiometric proportions. If thecoupling protein is added, the protein complex becomes capable ofhydroxylating 4-HPA to 3,4-dihydroxyphenylacetic acid. Thus, theoxidation of NADH,H⁺ is not wasted and allows the synthesis of ametabolite. The coupling protein alone has no enzyme activity.

(2) Prieto et al. (1993, 1994) and Xun & Sandvik (2000) suggest that the4-HPA 3-hydroxylase of E. coli W (ATCC 11105) is considered to be a newmember of the two-component flavin-diffusible monooxygenases (TC-FDM).The two components would be, firstly, 4-hydroxyphenylacetate3-hydroxylase, a 59 kDa monomeric enzyme encoded by the HpaB gene and,secondly, a 19 kDa monomeric flavin: NADH oxidoreductase encoded by theHpaC gene. In this case, FAD is reduced at the expense of NADH,H⁺ by theflavin: NADH,H⁺ oxidoreductase. The FADH₂ is then used by the oxygenaseto allow oxidation of the substrate using molecular oxygen.

The enzyme that we purified strongly oxidizes, NADH,H⁺ but produces verylittle homogentisate. In addition, the oxidation of NADH,H⁺ is dependenton 4-HPA being added. This suggests that we have an enzyme of the typeof that described by Prieto et al. We therefore consider that thepurified enzyme is the 4-HPA 1-hydroxylase (HPAH) sought. It is possiblethat, subsequently, it will be necessary to identify a coupling proteinin order to optimize the enzyme activity. The biochemical approach cantherefore be continued with the purified protein.

II.2.3—Production of the Internal Peptides and of the N-terminalSequence

The purified protein was sent to the Pasteur Institute to bemicrosequenced. Thus, the N-terminal sequence SHPAISLQAL RGSGADIQSIHIPYER (SEQ ID NO: 35) and six internal peptides named, respectively,peptides No. 11C, 12D, 20A, 22B, 23 and 24, as a function of the orderin which they leave the column: ATDFITPK (SEQ ID NO: 36), LGVGQPMVDK(SEQ ID NO: 37), VVFAGDSAHG VSPFX (SEQ ID NO: 38), VTALEPQAEG AL (SEQ IDNO: 39), IDFQLGWDAD PEEEK (SEQ ID NO: 40), LSVPATLHGS ALNTPDTDTF (SEQ IDNO: 41), were thus obtained. The amino acid (methionine or valine)normally corresponding to the initiation codon of the gene (ATG or GTG)is not found on the N-terminal sequence. Homology analyses in theprotein bases using the BLASTP algorithm do not make it possible toidentify homologous proteins. On the basis of the protein sequencesobtained, the corresponding degenerate oligonucleotides weresynthesized. These oligonucleotides were used in PCR reactions in orderto identify a portion of the gene encoding the purified and partiallysequenced HPAH protein.

II.2.4—Production of the PCR Fragment

PCR amplification of a portion (536 bp) of the gene encoding the 4-HPA1-hydroxylase was obtained using the degenerate primers Hy4R: TCYTCNGGRTCNGCRTCCCA (SEQ ID NO: 42) and Hy5F: GGNGTNGGNC ARCCNATGGT (SEQ ID NO:43) which encode, respectively, peptides 23 and 12D. These primers havea hybridization temperature of 55.4° C. and exhibit a degeneracy of 128and 512 respectively. The amplified sequence is cloned into the vectorpGEMT-easy and is then sequenced. Analysis of the sequence obtainedmakes it possible to find, besides the sequences encoding the peptidesHy4R and Hy5F, the nucleic acid sequence encoding internal peptide 22B.The latter element makes it possible to confirm that we have indeedamplified a portion of the gene encoding the purified HPAH protein. Atthis stage, homology searches in the protein bases, using the BLASTXalgorithm, bring up some weak homologies with hydroxylases, oxidases andmonooxygenases. Using the 536 bp PCR-amplified sequence, we will be ableto screen a P. acidovorans cosmid library in order to search for thecosmid containing the complete gene.

II.2.5—Screening of the P. acidovorans Cosmid Library

Screening of the cosmid library, using as probe the sequence obtainedabove, made it possible to identify 4 groups of cosmids considered to bedifferent on the basis of their restriction and hybridization profilesafter transfer by the Southern technique. Cosmids No. 1, 2 and 6 formthe first group, cosmids No. 3, 7 and 9 form the second, while cosmidsNo. 5 and 8 form the third. The final group is represented by cosmid No.4. The hybridization results suggest, in addition, that the hpaH genesought is present as a single copy in the genome of Pseudomonasacidovorans. We identified cosmids comprising at least a portion of thegene encoding the purified HPAH protein. In the meantime, we observedthat P. putida was incapable of growing on 4-HPA but could grow usingHGA as the only carbon source. This therefore gives us an excellentscreen for functional complementation; we will thus be able to definewhich of these cosmids comprises the functional gene encoding the 4-HPA1-hydroxylase activity.

II.2.6—Functional Complementation with the Cosmids

The nine cosmids previously identified are introduced into P. putida byelectroporation. The clones obtained are then subcultured on M63 mediumcontaining 4-HPA as the only carbon source. After 7-8 days, only thebacteria containing cosmid No. 8 succeeded in growing; that is to say,only cosmid. No. 8 contains all the expressible information allowingconversion of 4-HPA to HGA which can be used by P. putida. The cosmid isnamed Ccos8. The transformation with all the cosmids was repeated. Itwas always cosmid 8 which allowed complementation after a certain periodof time (6-10 days). In order to be able to move forward in our approachof determining the minimum DNA fragment expressing the 4-HPA1-hydroxylase activity, it is necessary to subclone Ccos8. The subcloneof interest is selected using the functional complementation screen.

II.2.7—Subcloning by Functional Complementation

Digestion of the cosmid with Not I makes it possible to obtain 6 DNAfragments of between 1.7 and 10 kb in size. These fragments weresubcloned into pBBR1MCS-Gm-Not-U. Five subclones of Ccos8 were obtained.Restriction analysis showed that the 4 and 10 kb fragments were notsubcloned. On the other hand, we observed that the 5 kb band initiallyobserved was in fact a double band of 5.1 and 5.2 kb. These clones werepassed, by triparenteral conjugation, from E. coli to P. putida. After 5days, only P. Putida containing the subclone corresponding to the 5.2 kbband of the cosmid Ccos8 grew on M63 containing 4-HPA as the only carbonsource. We had therefore just, identified the minimum fragmentcomprising the 4-HPA 1-hydroxylase activity. The clones corresponding tothe 5.2 kb are named 5 kbC. To confirm the result of the functionalcomplementation, we caused the plasmid p5kbC to be eliminated using thestrategy of incompatible origins of replication and forcing theelimination of the plasmid p5kbC, by selection pressure from theantibiotics used. We observed that P. putida lost the ability to grow on4-HPA as the only carbon source when it lost the plasmid p5kbC. Weconcluded therefrom that the 4-HPA 1-hydroxylase enzyme activity isclearly carried by the plasmid p5kbC. We could therefore have the 5.2 kbinsert sequenced, which should allow us to identify the functional hpaHgene.

II.2.8—Analysis of the 5.2 kb Sequence

The 5.2 kb insert of the plasmid p5kbC was sequenced. A nucleic acidhomology search (BLASTN) thus made it possible to identify threeportions in the insert. The first portion between bases No. 1 and 1465is completely homologous to a portion of the plasmid BirminghamIncP-alpha. It is therefore probably, a sequence derived from pLAFR5. Asecond nucleic acid portion between bases No. 1466 and 1695 exhibitscomplete homology with a portion of the cloning plasmid M13 mp8/pUC8.This sequence is therefore also part of pLAFR-5; specifically, themultiple cloning site of pLAFR-5 originates from pUC8 (Keen et al.,1988). Thus, the Eco RI and Sma I sites (FIG. 7) at respective positions1689 and 1695 are probably the cloning sites of pLAFR-5. The thirdportion, between bases 1696 and 5264 (i.e. 3568 bp), does not exhibitany strong homologies. This portion of DNA originates from the P.acidovorans genome. When the 5.2 kb sequence is analyzed using theBLASTX algorithm, probable proteins are identified (FIG. 7). Thus, theprotein encoded by gene 1 exhibits weak homologies with beta-lactamases,dehydrases and cyclases. The purified protein is encoded by gene 2 sincethe sequences encoding the internal peptides previously obtained arefound; it is therefore probably the 4-HPA 1-hydroxylase. The proteinalignments show that this protein exhibits some homologies withoxygenases and hydroxylases. The protein potentially encoded by gene 3exhibits no homologies with the databases. Finally, gene 4 probablyencodes an operon regulator.

A finer analysis of hpaH gene will now be made. According to theN-terminal protein sequence obtained, the ATG initiation codon of the4-HPA 1-hydroxylase protein is in fact 78 bp downstream of a GTGinitiator codon in phase with the ATG. The Shine-Dalgarno sequence AGGA,allowing ribosome binding, is found upstream of the initiator ATG butnot upstream of the GTG initiator codon, which confirms that the codingregion begins at the ATG initiator codon. The portion between the GTGand ATG codons probably does not therefore correspond to a preprotein.Thus defined, the hpaH gene is 1737 bp long and ends with the TGA stopcodon. The gene consists of 70.9% of GC bases.

Now that we have defined with precision the limits of the hpaH gene, theproduct of its translation: the HPAH protein, will be analyzed.

II.2.9—Analysis of the HPAH Gene

The hpaH sequence is translated using the universal codon system. A 563amino acid protein is thus obtained, which represents a molecular weightof 62.2 kDa. The protein homology searches (BLASTP) show that the HPAHexhibits approximately 15 to 25% identity essentially with proteins ofGram-positive organisms, encoding enzyme activities apparently verydifferent from that sought. Thus, a Streptomyces argillaceus oxygenase,E. coli 3-(3-hydroxyphenyl)propionate hydroxylase (EC 1.14.13.-),Sphingomonas sp. 2,4-dihydroxybenzoate monooxygenase, the enzymecatalyzing the 6-hydroxylation of tetracycline in Streptomycesaureofaciens, and a potential Streptomyces fradiae oxygenase are found.In fact, the HPAH exhibits homologies with the proteins of the phenolmonooxygenase (pheA) family and those of the 2,4-dichlorophenolhydroxylase (tfdB) family. The alignment corresponding to theabovementioned proteins is performed using the ClustalW algorithm (FIG.8). It makes it possible to demonstrate very conserved boxes. Threeunits of interaction with FAD are noted, inter alia. The first (GXGXXG)(SEQ ID NO: 44) corresponds to the β-α-β structural unit which allowsinteraction of the ADP component of FAD with the protein. The secondunit, (A/C)DG, is involved in the binding of FAD, while the third unit,G(R)VXX(A)GD(A)XH (SEQ ID NO: 45), allows interaction with the flavincomponent of FAD. Although the enzyme uses NADH,H⁺, the correspondingbinding site (GDH) is not identified. This absence of NADH,H⁺-bindingsite is a characteristic often observed in other FAD monooxygenases.Finally, a unit (DXXXLXWKLX XXXXXXXXXX LLXXYXXER) (SEQ ID NO: 46) isobserved which is also found in other hydroxylases (Ferrandez et al.,1997), but the meaning of which is not understood. Although the3-(3-hydroxyphenyl)propionate hydroxylase of E. coli catalyzes ahydroxylation reaction on a substrate structurally close to 4-HPA, theinformation acquired with this bioinformatic analyses does not make itpossible to be sure that we have indeed identified the 4-HPA1-hydroxylase. The only way to do this is to express the hpaH gene andto study its enzyme activity.

II.2.10.1—Expression of the hpaH Gene Encoding the 4-HPA 1-HydroxylaseActivity

In order to confirm that the hpaH gene encodes the 4-HPA 1-hydroxylaseactivity, it is necessary to express the gene. To do this, a two-stepcloning strategy is used, making it possible to eliminate genes No. 1and 3 and to place the hpaH gene under the control of the lac promoterof the original vector pBBR1MCS-Gm-Not-U. The plasmid obtained is namedpBBR1MCS FT12Δ1. A crude extract is produced from a culture, on richmedium, of P. putida transformed with this plasmid. The search foractivity by spectrophotometry (at 340 and 292 nm) shows that the clonedefinitely has the NADR,H⁺ oxidase activity induced by adding 4-HPA, butdoes not have the ability to synthesize homogentisate from the 4-HPA. Onthe other hand, the appearance of a molecule Z having a very closeretention time (tr=1.2 minutes versus 1.4 minutes) but a UV spectrumvery different from that of HGA. We put forward the hypothesis that HPAHoxidizes NADH,H⁺ so as to reduce its cofactor FAD. The reoxidation ofFAD takes place to the detriment of 4-HPA since it is the addition of4-HPA which initiates the reaction. The 4-HPA is therefore converted tometabolite Z. The UV spectrum of this metabolite suggests that the ringis no longer aromatic but may, however, be unsaturated. A structuralhypothesis for metabolite Z is presented in FIG. 2. This experimentshows that the lac promoter is functional in P. putida in the absence ofIPTG inducer, which suggests that the lad repressor is naturally absentin P. putida. We also demonstrate that the protein initially purified(HPAH) is really a 4-HPA-dependent NADH,H⁺ oxidase which converts 4-HPAto metabolite Z. The HPAH does not produce HGA. It is thereforenecessary to identify the partner protein(s) of this NADH,H⁺ oxidasedependent on 4-HPA, the addition of which makes it possible to restorethe 4-HPA 1-hydroxylase activity.

II.2.10.2—Identification of the HPAC Protein by Gel Filtration

We have seen that the 4-HPA 1-hydroxylase activity disappeared duringthe purification of the HPAH on a Red affinity column. We therefore putforward the hypothesis that the partner protein(s) of the4-HPA-dependent NADH,H⁺ oxidase were not retained by the Red 120 agaroseaffinity resin and are therefore recovered in the flow-through. Wetherefore decided to purify the flow-through and to search for theprotein(s) which, when added to the HPAH, made it possible to restorethe 4-HPA-1-hydroxylase activity. To do this, the flow-through isconcentrated by ultrafiltration (10K Macrosep™) and then loaded onto anS75 gel filtration column. A flow rate of 1 mL.min⁻¹ is applied and the1 mL fractions are collected. Enzyme reactions are then carried out,mixing together 50 μL of each fraction and 10 μL of HPAH purifiedbeforehand on a Red column, under normal reaction conditions. Thestopped reactions are then analyzed by HPLC. It is observed thatfractions 90 to 108, when added to HPAH protein, make it possible toproduce more metabolite Z. The production of metabolite Z is detected inthese same fractions in the absence of introduction of HPAH. Moreover,on the acrylamide gel corresponding to these fractions, we observe aprotein of molecular weight equivalent to HPAH. We concluded that theflow-through still contained a little HPAH protein. When fractions 109to 143 are added to the HPAH protein, the production of HGA is observed.The greater the production of HGA, the weaker the production ofmetabolite Z. The maximum production of homogentisate is obtained forfractions 116 to 128. Loading the fractions between 95 and 145 ontoacrylamide gel shows that a protein is highly enriched in fractions 109to 143, i.e. the chromatographic profile of this protein coincides withthe production profile of HGA. We decided to name this protein HPAC. TheHPAC protein is excised from the gel and then microsequenced at theN-terminal. The sequence obtained, MTTKTFA (SEQ ID NO: 47), shows thatthis protein is encoded by gene 1 (FIG. 7), which we henceforth namedbeginning at liC. This experiment shows that the 4-HPA 1-hydroxylaseactivity involves two proteins, HPAH and HPAC. However, we have notdefined the nature of the interaction between these two proteins: (1)are HPAH and HPAC both enzymes, or else (2) does HPAH have an enzymeactivity which is modifiable as a function of the interaction with HPAC.

II.2.10.3—Nature of the Interactions between HPAH and HPAC

The preceding experiment demonstrates that the HPAH and HPAC proteinsare necessary to reconstitute the 4-HPA 1-hydroxylase activity. Twohypotheses to explain the respective role of these proteins are putforward. In this paragraph, we present the results which suggest thatHPAC is an enzyme in its own right. Fractions 100, 101 and 102 from thegel filtration are pooled. They contain the HPAH, i.e. the NADH,H⁺oxidase activity which makes it possible to produce metabolite Z from4-HPA. Moreover, fractions 123, 124 and 125 from the gel filtration arepooled. They contain the HPAC. Various enzyme reactions are carried outusing the HPAH and/or the HPAC. These reactions are carried out in twosteps. A first reaction is carried out with the HPAH (respectivelyHPAC), and it is stopped after 30 minutes by heat treatment (100° C., 10min). The HPAC (respectively HPAH) is then added and the reaction ispursued for 30 minutes. The reaction is finally stopped by addingperchloric acid. Reactions are also carried out by replacing one of theenzymes with water. Finally, equivalent experiments are carried out byfiltering the reactions through 10 kD Nanosep™ (Pall Filtron) instead ofboiling them.

TABLE NO. 1 summarizes the results obtained Experiment Metabolite No.Enzyme No. 1 Enzyme No. 2 observed HPAH, HPAC / HGA A HPAH H₂Ometabolite Z B HPAH HPAC HGA C H₂O HPAC / D HPAC H₂O / E HPAC HPAHmetabolite Z F H₂O HPAH metabolite Z

We observe that the only way to produce the HGA is to have the twoproteins HPAH and HPAC simultaneously or successively in this order.When the HPAH is alone, or when the HPAC is introduced before the HPAH,only metabolite Z is detectable. Finally, the HPAC protein has no enzymeactivity on 4-HPA. These results suggest that metabolite Z is a reactionintermediate. The HPAH would convert 4-HPA to metabolite Z, thisreaction allowing the oxidation of NADH,H⁺. Metabolite Z would then beconverted to HGA by the HPAC. Physical interactions between the twoproteins do not appear to be necessary since the HPAH protein can bedenatured or removed by filtration before adding the HPAC. We haveshown, in vitro, that the 4-HPA 1-hydroxylase activity depends on theHPAC and HPAH protein. However, the HPAC protein is not pure on exitingon gel filtration, it is only enriched. It therefore remains possiblethat, in reality, it is another protein contained in this enrichedextract which converts metabolite Z to HGA. In order to eliminate thedoubts, we decided to clone the two genes (hpaC and hpaH) on the samevector; in this case, we should produce the 4-HPA 1-hydroxylase activityand therefore be able to make P. putida grow on minimum mediumcontaining 4-HPA as the only carbon source.

II.2.10.4—Functional Complementation of P. putida with hpaH and hpaC

The plasmid pL1lac2 (FIG. 9) is a vector pBBR1MCS-Gm^(R) containing thehpaC gene under the control of the P. fluorescens HPPD promoter and, inthe opposite direction, the hpaH gene under the control of a lacpromoter. The plasmid is introduced into P. putida by electroporation.The bacteria are then plated out on minimum medium containing or notcontaining 4-HPA as the only carbon source. After 5 days, the coloniesare visible only on dishes containing 4-HPA as the only carbon source.After 8 days, the colonies are a good size. The plasmid DNA extractedfrom these colonies confirms the presence of the whole plasmid pL1lac2.Moreover, P. putida is incapable of growing on 4-HPA when the bacteriumis transformed with the vector pBBR1MCS-GMR containing either the hpaCgene or the hpaH gene. The functional complementation obtained in thisexperiment confirms that the hpaC and hpaH genes are necessary andsufficient to initiate the 4-HPA 1-hydroxylase activity sought.

EXAMPLE III Construction of the Various Cytosolic Expression Cassettes

III.1—HPAC

The HPAC gene was isolated from Pseudomonas acidovorans by PCR on aplasmid derived (p5kbC) from a genomic DNA cosmid library, using thefollowing oligonucleotides:

(SEQ ID NO: 48) Start ORF1 (AflIII): GCAGGATGCA CATGTCCACC AAGAC (SEQ IDNO: 49) ORF1 Fin (HindIII): CGGACGCAAG CTTGCATCAG CCTTC

The reaction was carried out according to standard conditions. Theamplified fragment, 993 bp in size, was subcioned into the plasmidpGEMTeasy (Promega) according to the supplier's protocol. The plasmidpOZ150 thus obtained was sequenced. The cassette obtained by EcoRI+SpeIdigestion was cloned into the plasmid pBluescriptII-KS+opened with thesame enzymes, to give the plasmid, pEPA13. The CsVMV promoter isisolated from the plasmid pCH27, derived from the plasmid pUC19containing the expression cassette for a herbicide tolerance gene underthe control of CSVMV. For this, a standard PCR was carried out on athermocycler with Pfu polymerase generating blunt ends; 1 cycle of 5 minat 95° C., 30 cycles [95° C. 30 sec, 57° C. 30 sec, 72° C. 1 min], 72°C. 3 min. The primers used are: N-CsVMV: GCCCTCGAGG TCGACGGTATTGATCAGCTT CC (SEQ ID NO: 50) introducing the XhoI and BclI sitesC-CsVMV: CGCTCTAGAA TTCAGATCTA CAAAC (SEQ ID NO: 51) (EcoRI)

The 565 bp fragment generated is digested with XhoI+EcoRI before beinginserted into the plasmid pEPA13 digested beforehand with XhoI+EcoRI;the plasmid pEPA14 is obtained. The Nos terminator is isolated from theplasmid pRD11, derived from pBlueScript II-SK(−) in which the Nosterminator is cloned, by HindII+NotI digestion. The 292 bp fragmentobtained is cloned into the plasmid pEPA14 opened with the same enzymes,giving pEPA15.

pEPA15 cassette=CsVMV promoter-hpa C-Nos terminator (FIG. 10; SEQ ID No.19).

III.2. HPAH

The HPAH gene was isolated from Pseudomonas acidovorans by PCR on aplasmid derived (p5kbC) from genomic DNA cosmid library, using thefollowing oligonucleotides:

-   -   Start ORF2 (AflIII): CAGAGGACGA ACAACATGTC CCACC (SEQ ID NO: 52)    -   ORF2 Fin3 (HindIII): CTGTGGATGA AGCTTAAGAG GTTCAGGC (SEQ ID NO:        53)

The reaction was carried out according to standard conditions. Theamplified fragment, 1729 bp in size, was subcloned blunt-ended into theplasmid pBlueScript II SK digested with EcoRV. The plasmid pEPA16 thusobtained was sequenced. The CaMV 35S promoter is isolated from theplasmid pCH14, derived from the plasmid pBI 121 containing the GUSexpression cassette: CaMV 35S promoter-GUS-Nos terminator. For this, astandard PCR was carried out on a thermocycler with Pfu polymerasegenerating blunt ends; 1 cycle of 5 min at 95° C. 30 cycles [95° C. 30sec, 63° C. 30 sec, 72° C. 1 min], 72° C. 3 min. The primers used are:

-   N-CaMV: GCATGCCTCG AGCCCACAGA TGG (SEQ ID NO: 54) introducing the    XhoI site-   C-CaMV: CCACCCGGGG ATCCTCTAGA G (SEQ ID NO: 55) introducing the    BamHI site.

The 839 bp fragment generated is digested with XhoI+BamHI before beinginserted into the plasmid pEPA16 digested beforehand with XhoI+BclI: theplasmid pEPA17 is thus obtained. The Nos terminator is isolated from theplasmid pRD11 by PCR, under the same conditions as previously, for 1cycle of 5 min at 95° C., 30 cycles [95° C. 30 sec, 57° C. 30 sec, 72°C. 1 min], 72° C. 3 min, with the following primers:

-   N-Nos: CAAGCTTATC GATACCGTCG ACG (SEQ ID NO: 56) introducing HindIII-   C-Nos: GSSTTGCGGC CGCAATTCCC GACCTAGGA ACATAG (SEQ ID NO: 57)    introducing-NotI an AvrII.

The 305 bp fragment obtained is digested with NotI+HindIII before beingcloned into the plasmid pEPA17 opened with the same enzymes, givingpEPA18.

pEPA18 cassette=CaMV 35S promoter-hpaH-Nos terminator (FIG. 11; SEQ IDNo. 17).

III.3 HPPO

The HPPO gene was isolated from Arthrobacter globiformis by PCR on thecosmid 2A derived from a genomic DNA cosmid library, using the followingoligonucleotides:

-   N-term-HPPO-ScaI: GAATTCAGTA CTTCACTTAC AGTGTCCGGC (SEQ ID NO: 58)    introducing the EcoRI and ScaI restriction sites;-   C-term-HPPO-AsuII-XhoI: GAATTCTCGA GTTCGAACAA ACTGAGTAGC AGCTCA (SEQ    ID NO: 59) introducing the EcoRI, XhoI and AsuII sites.

The reaction was carried out according to standard conditions. The 1800bp fragment obtained is cloned into the vector pGEMT-easy (Promega)according to the supplierts protocol. The plasmid pOZ151 thus obtainedwas sequenced. The cassette obtained by digestion with SphI+XhoI wascloned into the plasmid pBBR1-MCS (Gm) opened with the same enzymes, togive the plasmid pEPA20. The histone simple promoter is isolated fromthe plasmid pCH9, derived from the plasmid pUC19 containing theexpression cassette for EPSPS: histone simplepromoter-intron2-OTP-EPSPS-histone terminator. For this, a standard PCRwas carried out with Pfu polymerase generating blunt ends; 1 cycle of 5min at 95° C., 5 cycles [95° C. 30 sec, 45° C. 30 sec, 72° C. 1 min], 30cycles [95° C. 30 sec, 65° C. 30 sec, 72° C. 1 min], 72° C. 3 min. Theprimers used are:

-   N-SH: GCTTGCATGC CTAGGTCGAG GAGAAATATG (SEQ ID NO: 60) introducing    the SphI and AvrII sites-   C-SH: CATGAGGGGT TCGAAATCGA TAAGC (SEQ ID NO: 61)

The 970 bp fragment generated is digested with SphI before beinginserted into the plasmid pEPA20 digested beforehand with SphI+ScaI; inthe plasmid pEPA21 obtained, the initiating ATG of the HPPO gene isrecreated behind the simple histone promoter. The histone terminator isisolated from the same plasmid pCH9 by PCR, under the same conditions aspreviously, for 1 cycle of 5 min at 95° C., 35 cycles [95° C. 30 sec,55° C. 30 sec, 72° C. 1 min], 72° C. 3 min, with the following primers:

-   N-Hister: CTAGACCTAG GGGATCCCCC GATC (SEQ ID NO: 62) introducing    AvrII-   C-Hister: CCCACTAGTG TTTAAATGAT CAGTCAGGCC GAAT (SEQ ID NO: 63)    introducing SpeI and BclI.

The 726 bp fragment obtained is digested with SpeI+AvrII before beingcloned into the plasmid pEPA21 opened with SpeI, giving pEPA22.

pEPA22 cassette=histone simple promoter-hppO-histone terminator (FIG.12; SEQ ID No. 15).

III.4. Association of the Genes

The cassette containing the HPAC gene is extracted from pEPA15 by NotIdigestion and cloned into pEPA18 (NotI+Bsp120I) so as to form pEPA19(FIG. 13; SEQ ID No. 21). The latter is digested with AvrII so as toclone the extracted cassette into the AvrII+SpeI sites of pEPA22. Theplasmid containing the three constructs is pEPA23 (FIG. 14; SEQ ID No.22).

III.5. Binary Vector

In order to transform the plants with Agrobactetium, the threeconstructs can be extracted with BclI in order to be introduced into abinary vector of Agrobacteria.

Abbreviations: 3,4-DHPA 3,4-dihydroxyphenylacetic acid 4-HPA4-hydroxyphenylacetic acid DNA deoxyribonucleic acid APcI AtmosphericPressure chemical Ionization RNA ribonucleic acid mRNA messengerribonucleic acid ETB ethidium bromide BLAST Basic Local Alignment SearchTool BSA bovine serum albumin C¹⁰⁰ carbenicillin (100 ?g/mL) CRLD Centrede Recherche La Dargoire [La Dargoire Centre for Research] Da Dalton DKNisoxaflutole diketonitrile DMSO dimethyl sulfoxide dATP2′-deoxyadenosine 5′-triphosphate dCTP 2′-deoxycytidine 5′-triphosphatedGTP 2′-deoxyguanosine 5′-triphosphate dNTP 2′-deoxynucleotide5′-triphosphate dTTP 2′-deoxythymidine 5′-triphosphate DTEdithioerithritol DTT 1,4-dithiothreitol EDTA ethylenediaminetetraaceticacid FAD flavin adenine dinucleotide FPLC fast protein liquidchromatography Gm²⁰ gentamycin (20 ?g/mL) HGA homogentisic acid HPLChigh performance liquid chromatography HPP hydroxyphenylpyruvic acidHPPD hydroxyphenylpyruvic acid dioxygenase HPPO hydroxypherlylpyruvateoxidase IFT isoxaflutole IPTG isopropyl-?-thiogalactopyranoside Kn⁵⁰kanamycin (50 ?g/mL) kb kilo bases Km Michaelis Menten constant L-DOPA3,4-dihydroxyphenylalanine LB Luria Bertani medium min minutes mJmillijoules MNDD manganese dependent dioxygenase MndD gene encoding MNDDNAD⁺ (H, H⁺) nicotinamide adenine dinucleotide (oxidized form/reducedform) GMO genetically modified organism OTP optimized transit peptide bpbase pairs pBBR1MCS-Gm plasmid pBBR1MCS resistant to gentamycin PCRpolymerase chain reaction ppm parts per million; mg.L⁻¹ PVDFpolyvinylidene difluoride qs quantity sufficient for Q.r. respiratorycoefficient Rif¹⁰⁰ rifampicin (100 ?g/mL) NMR nuclear magnetic resonanceSDS sodium dodecyl sulfate sec second TBE trisborate EDTA TEV tobaccoetch virus TEA trifluoroacetic acid TrEMBL translated EMBL bank Tristris(hydroxymethyl)aminomethane U.V. ultraviolet vs versus X-gal5-bromo-4-chloro-3-?-D-galactopyranoside

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1. An isolated nucleic acid encoding an HPP oxidase, characterized inthat it comprises a nucleic acid sequence selected from the groupcomprising the coding sequences of SEQ ID No. 1, SEQ ID No. 3, SEQ IDNo. 5 and SEQ ID No. 15, or a nucleic acid capable of selectivelyhybridizing to the coding sequence of said SEQ ID No. 1, 3, 5 or 15under the following conditions: prehybridization for four hours at 68°C. in a solution of 5×SSC, 6% Denhardt's, and 0.5% SDS; hybridizationfor sixteen hours at 68° C.; and washes at 65° C. in 0.5×SSC, 0.1% SDS.2. An expression cassette comprising a coding sequence, characterized inthat the coding sequence comprises the nucleic acid as claimed inclaim
 1. 3. A cloning or expression vector, characterized in that itcomprises the expression cassette as claimed in claim
 2. 4. Atransformed plant cell, characterized in that it comprises theexpression cassette as claimed in claim
 2. 5. A transformed plant,characterized in that it comprises the expression cassette as claimed inclaim
 2. 6. A seed of a transformed plant as claimed in claim 5,characterized in that it comprises at least one the expression cassettecomprising a coding sequence, characterized in that the coding sequencecomprises a nucleic acid sequence which encodes a polypeptide that hasHPP oxidase activity.
 7. A method of transforming plants, characterizedin that the expression cassette as claimed in claim 2 is introduced intotheir genome.
 8. A method for selective weeding of plants comprising thestep of applying an HPPD inhibitor to a transformed plant as claimed inclaim
 5. 9. A method of weed killing in an area of a field comprisingthe seed as claimed in claim 6, characterized in that it comprises theapplication, in said area of the field, of a dose, which is toxic forsaid weeds, of an HPPD-inhibiting herbicide, without howeversubstantially affecting the seed.
 10. A method of growing a transformedplant as claimed in claim 5, characterized in that it comprises sowing aseed of said transformed plant in an area of a field suitable forgrowing said plant, applying to said area of said field a dose, which istoxic for the weeds, of a herbicide having HPPD as the target, in theevent of weeds being present, without substantially affecting said seedor said transformed plant, then harvesting the plants grown when theyreach the desired maturity and, optionally, separating the seeds fromthe harvested plants.
 11. The method as claimed in claim 8,characterized in that the herbicide is applied in pre-emergence and/orin post-emergence.
 12. A method of weed killing in an area of a fieldcomprising a transformed plant as claimed in claim 5, characterized inthat it comprises the application, in said area of the field, of a dose,which is toxic for said weeds, of an HPPD-inhibiting herbicide, withouthowever substantially affecting the transformed plant.
 13. The method asclaimed in claim 9, characterized in that the herbicide is applied inpre-emergence and/or in post-emergence.
 14. The method as claimed inclaim 10, characterized in that the herbicide is applied inpre-emergence and/or in post-emergence.
 15. A method of growing plantstolerant to a herbicide which is an HPPD inhibitor, comprising the stepof growing plants expressing at least one enzyme insensitive to saidherbicide that allows conversion of para-hydroxyphenylpyruvate (HPP) to4-hydroxyphenyl-acetic acid (4-HPA) and at least one enzyme insensitiveto said herbicide that allows conversion of 4-HPA to homogentisate as aresult of nucleic acid encoding said enzymes being inserted into thegenome of said plants, wherein said at least one enzyme that allowsconversion of para-hydroxyphenylpyruvate (HPP) to 4-hydroxyphenyl-aceticacid (4-HPA) is HPP oxidase, wherein said HPP oxidase comprises an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6and
 16. 16. The method as claimed in claim 15, characterized in that anHPAH and an HPAC are expressed in the plant.
 17. The method of claim 15wherein said HPP oxidase is encoded by the coding sequence of a nucleicacid selected from the group consisting of SEQ ID NOS: 1, 3, 5 and 15.18. A method of growing plants tolerant to a herbicide which is an HPPDinhibitor, comprising the step of growing plants expressing at least oneenzyme insensitive to said herbicide that allows conversion ofpara-hydroxyphenylpyruvate (HPP) to 4-hydroxyphenyl-acetic acid (4-HPA)as a result of nucleic acid encoding said enzyme being inserted into thegenome of said plants, wherein said at least one enzyme that allowsconversion of para-hydroxyphenylpyruvate (HPP) to 4-hydroxyphenyl-aceticacid (4-HPA) is HPP oxidase, wherein said HPP oxidase comprises an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6and
 16. 19. The method of claim 18 wherein said HPP oxidase is encodedby the coding sequence of a nucleic acid selected from the groupconsisting of SEQ ID NOS: 1, 3, 5 and
 15. 20. A method for making plantstolerant to a herbicide which is an HPPD inhibitor, comprising the stepsof inserting a nucleic acid encoding at least one enzyme insensitive tosaid herbicide that allows conversion of para-hydroxyphenylpyruvate(HPP) to 4-hydroxyphenyl-acetic acid (4-HPA) and a nucleic acid encodingat least one enzyme insensitive to said herbicide that allows conversionof 4-HPA to homogentisate, wherein said at least one enzyme that allowsconversion of para-hydroxyphenylpyruvate (HPP) to 4-hydroxyphenyl-aceticacid (4-HPA) is HPP oxidase, and wherein said HPP oxidase comprises anamino acid sequence selected from the group consisting of SEQ ID NOS: 2,4, 6 and 16, into the genome of a plant cell; regenerating a plant fromsaid plant cell; and growing said plant, wherein said at least oneenzyme insensitive to said herbicide that allows conversion ofpara-hydroxyphenylpyruvate (HPP) to 4-hydroxyphenyl-acetic acid (4-HPA)and said at least one enzyme insensitive to said herbicide that allowsconversion of 4-HPA to homogentisate are expressed in said plant.
 21. Amethod for making plants tolerant to a herbicide which is an HPPDinhibitor, comprising the steps of inserting a nucleic acid encoding atleast one enzyme insensitive to said herbicide that allows conversion ofpara-hydroxyphenylpyruvate (HPP) to 4-hydroxyphenyl-acetic acid (4-HPA),wherein said at least one enzyme that allows conversion ofpara-hydroxyphenylpyruvate (HPP) to 4-hydroxyphenyl-acetic acid (4-HPA)is HPP oxidase, and wherein said HPP oxidase comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 2, 4, 6 and16, into the genome of a plant cell; regenerating a plant from saidplant cell; and growing said plant, wherein said at least one enzymeinsensitive to said herbicide that allows conversion ofpara-hydroxyphenylpyruvate (HPP) to 4-hydroxyphenyl-acetic acid (4-HPA)is expressed in said plant.